Claims:

1. A method for identifying a human gene sequence useful for developing an
anti aging pharmaceutical product or a pharmaceutical product for
treating aging related disease, comprising:obtaining Xist gene sequences
of a population of long-living individuals; andcorrelating the Xist gene
sequences to sex and age dependent characteristics, wherein sequences
having high correlation to sex and old-age define template sequences for
designing said pharmaceutical products.

2. The method of claim 1, wherein the population consists of individuals
at least 80 years or older.

3. The method of claim 1, wherein the population consists of individuals
at least 100 years or older.

4. The method of claim 1, wherein the population comprises individuals
suffering from aging related diseases.

6. The method of claim 1, wherein the pharmaceutical product comprises an
Xist RNA having a sequence identified from the correlation step,
alternate splice forms of the sequence, or fragments thereof.

7. A method for manipulating the aging process or treating an aging
related disease, comprising:administering a pharmaceutically active
composition comprising a Xist RNA, alternate splice forms of the RNA, a
fragment thereof, or an analog thereof to a subject.

8. The method of claim 7, wherein the Xist RNA has a sequence
substantially homologous to the Xist RNA sequence of a long-living
healthy individual.

10. The method of claim 7, wherein the administering step is by way of
direct injection or oral ingestion.

11. The method of claim 7, wherein the administering step is by way of a
biological vector engineered to produce Xist RNA in the subject, wherein
the vector is one selected from a bacteria, a virus.

12. The method of claim 11, wherein the vector is capable of tissue
specific targeting.

13. A method for treating aging or aging related diseases,
comprising:administering to a subject an agent capable of activating the
expression of Xist gene in the subject.

14. The method of claim 13, wherein the agent is one selected from a
nucleic acid, a polypeptide, a protein, a nucleic acid mimetic, a small
organic molecule, or a combination thereof.

15. The method of claim 14, wherein the nucleic acid is a Xist RNA or
fragments thereof.

16. The method of claim 15, wherein the agent is 5-azacytidine or an
analog thereof.

17. A method for in vivo evolution of genes to obtain genes having desired
properties, comprising:selecting a starting set of genes with high rates
sequence variation, wherein at least one gene is capable of cleaving
itself at a predetermined rate;replicating the genes via in vivo
replication to evolve the genes; andtesting the properties of the genes
at predetermined intervals for the desired property.

22. The kit of claim 18, wherein the vectors are provided in the form of a
library.

23. The kit of claim 18, wherein the gene sequences are sequences obtained
from long-living individuals.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims an invention which was disclosed in
Provisional Application No. 60/811,899 filed Jun. 7, 2006, entitled
"SEX-SPECIFIC REGULATION OF AGING AND APOPTOSIS". The benefit under 35
USC §119(e) of the United States provisional application is hereby
claimed. The above priority applications are hereby incorporated herein
by reference.

SEQUENCE LISTING

[0002]The present invention contains sequence listing.

FIELD OF THE INVENTION

[0004]The invention pertains to the field of senescence and evolution.
More particularly, the invention provides mechanism for sex-specific
regulation of aging and apoptosis.

BACKGROUND OF THE INVENTION

[0005]Recent and classic observations suggest that the mitochondria has an
important and ancient role in determining the distinction between
germ-line and soma, as well as sexual identity. In this regard, the fact
that the mitochondria genome is asymmetrically inherited is perhaps THE
defining feature of the oocyte.

[0006]The asymmetric inheritance feature of mitochondrial genes and sex
chromosome genes promotes the evolution of sexually antagonistic gene
functions (i.e. compromised gene function in one sex or both). The
present invention is based on the premises that such genes will
contribute preferentially to the aging phenotype.

SUMMARY OF THE INVENTION

[0007]Genetic analysis of Drosophila, mice and humans indicates that gene
alleles, mutations and transgenes that affect life span tend to have
different effects in an organism depending on the sex of the organism.
The likely reason for this is that the sexes have different genotypes
(e.g., X/X vs. X/Y) and face quite different environmental pressures
(e.g., to reproduce, males have to mate with females and vice versa, but
mate selection criteria for each sex may be very different). That is to
say, genes are subject to different genetic interactions and different
gene-by-environment effects in males than in females. The consequence is
that through evolution certain genes are differentially selected and
optimized for one sex over the other. The mitochondrial genome and the X
chromosome are such differentially selected genes.

[0008]Both the mitochondrial genome and the X chromosome are
asymmetrically inherited in Drosophila and mammals. Through evolution,
the mitochondrial spend relatively more time under selection in females
and are therefore expected to be better optimized for function in the
female than in the male. This hypothesis is supported by the fact that
the Drosophila X chromosome is a hotspot for sexually antagonistic
fitness variation.

[0009]In terms of the aging phenotype, Drosophila and mammals females tend
to live longer than males. This may be due in part to sub-optimal
mitochondrial function in males. One finds evidence for this hypothesis
in the observation that old Drosophila and old mammals exhibit
apoptosis--an observation that is consistent with the idea that
mitochondria are less functional during aging due to maternal-only
inheritance.

[0010]Together, these data support the conclusion that a significant part
of the aging phenotype is due to antagonistic pleiotropy of gene function
between the sexes.

[0011]With these considerations, the inventor of the present has devised a
molecular model which describes the co-regulation of sex determination,
apoptosis and life span based on the on/off status of a single gene: Sxl
in Drosophila melanogaster and Xist in humans (exemplary sequence of Sxl
is provided in SEQ ID:1-2, and Xist in SEQ ID 3). In accordance with on
this model, the present invention provides methods and products for
utilizing the of/off mechanism in a subject to effect an aging related
change in a subject.

[0012]In particular, one object of the present invention is to provide
anti-apoptotic agents and therapies involving human Xist gene, Xist RNA,
Xist gene product, antagonists of the above-mentioned nucleic acids and
proteins, and small molecule mimics of the above-mentioned nucleic acids
and proteins. Another object of the present invention is to provide
prophylactic anti-aging agents and therapies involving human Xist gene,
Xist RNA, Xist gene product, antagonists of the above-mentioned nucleic
acids and proteins, and small molecule mimics of the above-mentioned
nucleic acids and proteins.

[0013]A further object of the invention relates to the use of the finite
half-life gene segregation mechanism to produce in vitro evolution of
genes and the directed evolution of genes with desired properties.

[0014]The above-mentioned and other features of this invention and the
manner of obtaining and using them will become more apparent, and will be
best understood, by reference to the following description, taken in
conjunction with the accompanying drawings. The drawings depict only
typical embodiments of the invention and do not therefore limit its
scope.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 Asymmetric segregation of the X and Y chromosome and
mitochondrial genome (M). Drosophila and human males in generation 1 do
not pass mitochondrial genes (M) on to their offspring in generation 2,
rather these genes come from the female parent (maternal inheritance).
Asymmetric segregation or maternal inheritance of the mitochondrial
genome means the female gamete or egg contributes the functional
mitochondrial genomes to the embryo while the male gamete or sperm does
not. A, autosomes; M, mitochondrial genomes. SG=Switch Gene and
Sex-determination Gene.

[0019]FIG. 5 Invasion of the eukaryotic cell by the mitochondria. When the
mitochondria (M) invaded the eukaryotic cell it created competition for
inheritance between the mitochondria and the nucleus (N). The only way
the M could be maintained is if it provided some advantage to the cell.
In turn, the only way M could be maintained as an entity separate from N
is to have a finite half life, i.e., be lost at some rate by segregation
or apoptosis--this is the same thing as asymmetric segregation. The
simplest way to accomplish this is two states in N, one state that
prevents M loss, and one that does not.

[0020]FIG. 6 Genes and replicators. Gene A encodes a single-subunit
replicator that replicates gene A. A perfectly identical and symmetric
copy of gene A has no selective advantage because it encodes the same
replicator (by definition). One way a second gene B (such as an imperfect
copy of A) can be maintained as a separate entity is if it has a shorter
half-life than A (i.e., is lost at some rate). By definition this creates
two states for A: A alone and A+B, which is the same thing as asymmetric
segregation. To be maintained A+B must encode a better replicator (i.e.,
have greater fitness).

[0023]FIG. 9A-H Conditional over-expression of wild-type and
dominant-mutant p53 transgenes and Baculovirus p35 transgenes using
Geneswitch system. A-E. p53 transgene over-expression and controls. E-H.
Baculovirus p35 over-expression and controls. In FIG. 9E, Female
Larvae+N=3 lived 86, 92, 96 days respectively, with average 91.33 days.
Even though the p53WT line is very sick, there should be about 100 males
and 100 females/bottle totally, similar to the number of Cyo (not
carrying p53WT TG) offspring. But only 3 females came out from more than
10 bottles (12 or 14 bottles). Counting results attached. In FIG. 9F,
estimate # Male Larvae+ should have at least 80 mals/day coming out
(Or-R). But only 40/bottle at most was observed. There might be reduction
in Female numbers, too. In FIG. 9G, estimated # Male larvae+should have
at least 80 mals/day coming out (Or-R). But only 47/bottle at most was
observed. There might be reduction in Female numbers, too. In FIG. 9H,
estimated # Male Larvae+ should have at least 80 mals/day coming out
(Or-R). But only 27/bottle at most was observed. Females came out
76/bottle/day at most, less than Or-R (136/bottle/day). In all of FIG. 9,
UAS-p53WT, UAS-p35 CHX, CH2, CH3 lines all showed late eclosion; lots of
pupa seemed dead. UAS-p35 CH3 eclosed slower and less than UAS-p35 CHX,
CH2. Almost all pupa from p53WT failed to eclose (only 3 female escapers,
died on D86, D92, D96) UAS-p35 CHX, CH2, CH3 lines have lower male/female
ration than others (Or-R M/F>1/2).

[0024]FIG. 10 Diagram of transgenic constructs. (A) The "Tet-on"
conditional transgene expression system. The rtTA transgenic construct
(or "driver") contains the tissue-general actin5C promoter driving
expression of the artificial transcription factor rtTA. The target
constructs were generated by cloning the indicated cDNA fragments
downstream of the DOX-inducible promoter in the USC1.0 vector. The rtTA
protein will bind to the 7 Tet-O sites in the target construct promoter
and activate transcription only in the presence of DOX. (B) Diagram of
the sequence and reading frames of the hUbbWT and hUbb+1 constructs.
The number 1 indicates the A of the normal ATG start codon for
translation of hUbbWT. Note that any translation framed from position 2
(the T of the ATG start codon; or +1 reading frame) can produce the
antigenic peptide (indicated by red asterisk), followed by a stop codon
at position 41. The amino acid sequence of the peptide used to generate
the hUbb+1 antibody is indicated. (C) Diagram of the sequence and reading
frames of the hAppWT and hApp+1 constructs. The GAGAG hotspot is
located in hApp exon 9. The amino acid sequence of the peptide used to
generate the hApp+1 antibody is indicated. The transgenic strains
are given names designed to be informative, and include the name of the
inserted construct (e.g., hUbbWT or hUbb+1), the chromosome of
insertion in parentheses (e.g., chromosome 2 or 3 or X), and a unique
number indicating the independent insertion event. For example,
hUbbWT(2)[118] is an insertion of the hUbbWT construct on the second
chromosome, independent insertion event designated "118".

[0025]FIG. 11 Northern and Western analysis of conditional transgene
expression. Flies of the indicated genotypes were cultured for one week
on food supplemented +/-DOX, as indicated. A-C. Northern analysis. Total
RNA was isolated from 30 flies, quantified by spectrophotometer, and 5
μg (1×) and 10 μg (2×) amounts were loaded for each
sample. The resultant blot was hybridized with the indicated
gene-specific probes. A. Control flies and hUbbWT transgenic fly strains.
B. hAppWT transgenic fly strains. C. hUbb+1 transgenic fly strain.
D, E. Western analysis. Total protein was isolated from 30 male flies,
diluted as indicated, fractionated using SDS-PAGE and Western blotted. D.
Control and hUbbWT transgenic strain fly protein incubated with antibody
specific for hUbb. E. Control and transgenic strain fly protein incubated
with antibody specific for hApp.

[0026]FIG. 12 Western blot analysis using antibody specific for
hUbb+1. Total protein was isolated from 30 male flies of the
indicated genotypes, and 1/8 of the sample was assayed for the presence
of protein that would be recognized by hubb+1 antibody. Where
indicated protein samples were diluted 1:2, 1:3, 1:5 or 1:10 to confirm
sensitivity of the assay to relative protein concentrations. In panels
B-F all samples are diluted 1:3. A. Molecular weight markers were run
alongside His-tagged hubb+1 purified from E. Coli cells as well as
total protein isolated from 30 "young" (10 day old) and "old" (65 day
old) male Or-R control flies, as indicated. B. "Young" (10 day old) flies
of the indicated genotypes. C. Flies cultured +/- DOX for 26 days. D.
Flies cultured +/- DOX for 48 days. E. Flies cultured +/- DOX for 67
days. F. Flies cultured +/- DOX for 82 days. Where visible the gel
protein front (F) is also indicated. Solid arrowheads indicate two
species of <20 Kd, either of which might represent Ubb+1 monomer,
which has an expected size of ˜11 kd. Open arrowhead indicates
species at expected position for Ubb+1 ligated to one Ubb wild-type
protein (˜11 Kd+˜8.5 Kd=˜19.5 Kd). Single asterisk
indicates species at expected position for Ubb+1 ligated to two Ubb
proteins (˜11 Kd+˜17 Kd=˜28 Kd). Double asterisk
indicates species at expected position for Ubb+1 ligated to three Ubb
proteins (˜11 Kd+˜25.5 Kd=˜37 Kd). Estimations of sizes
of various species are presented in Supplemental Materials.

[0027]FIG. 13 Western blot analysis using antibody specific for
hApp+1. Total protein was isolated from 30 flies of the indicated
genotypes, and assayed for the presence of protein that would be
recognized by hApp+1 antibody; "young" is 10 days old and "old" is
65 days old. A. Molecular weight markers were run alongside His-tagged
hApp+1 purified from E. coli cells, as well as the indicated
dilutions of total protein isolated from flies in which the hApp+1
transgenic construct was expressed. B. Purified His-tagged hApp+1
protein from E. coli was run alongside protein from young and old Or-R
control flies. C. Flies cultured +/- DOX for 26 days. D. Flies cultured
+/- DOX for 48 days.

[0028]FIG. 14 Phenotypes of hUbb and hUbb+1 over-expression. A.
Frequency of adult flies of the indicated genotypes emerging from crosses
where larval development was allowed to occur in the presence and absence
of DOX, as indicated. In these experiments a threetransgene configuration
was used to achieve tissue-general, DOX-dependent expression. The
daughterless-Gal4 driver (Da-Gal4) yields tissue general expression of
the yeast transcription factor Gal4. The Gal4 protein activates
expression of the "901" bridge construct encoding rtTA[M2alt] under the
control of a UAS-promoter. In the presence of DOX, the rtTA will bind to
the TetO sites in the target construct and activate expression of the
transgene. Therefore DOX-dependent transgene expression can occur only in
progeny that inherit all three constructs (blue bars). In the crosses to
either Or-R wild-type or w[1118] flies, the target construct chromosome
is replaced by a wild-type chromosome, thereby controlling for the
effects of DOX itself. Arrows indicated reduced frequency of
hUbbWT-expressing flies emerging due to DOX-dependent pupal lethality. B.
Examples of pupal-lethal phenotype resulting from hUbbWT(70) transgenic
line cultured +/- DOX, as indicated. C-F. Life span assays. Flies
containing the indicated target construct insertions along with the
Da-Gal4 driver and "901" bridge construct, as well as controls were
generated as described above (A). The survival of flies of the indicated
genotypes was assayed +/- DOX, as indicated. The Or-R control data and
w[1118] control data is the same in each panel.

[0029]FIG. 15 Transgenic Control-GFP reporter and MM-GFP reporter. A.
Diagram of the Control-GFP and MM-GFP reporters. B. Flies containing the
Control-GFP reporter and rtTA(3)E2 driver cultured for one week +/- DOX,
as indicated. C. Flies containing the indicated MM-GFP reporter insertion
and rtTA(3)E2 driver cultured +DOX were irradiated as 4-day old adults,
or transferred to 100% oxygen atmosphere, and then photographed at the
indicated number of days after treatment. Shown for each fly are an
overlay of visible and GFP images, as well as the corresponding GFP image
alone.

DETAILED DESCRIPTION

[0030]As set forth above, it is an unexpected discovery of the present
invention that there exist an on/off switch mechanism at the
genetic/molecular level for controlling the life span of an organism. In
particular, it is an discovery of the present invention that the Xist
gene in humans and the Sxl gene in Drosophila act as regulators for
apoptosis and life span in the respective organism. In accordance with
the discoveries of the present invention, there are provided methods for
altering the aging process and for treating aging related diseases by
manipulating the genes and gene products of these two genes and their
respective host organisms.

[0031]While not intending to be limiting in any way, the following
theoretical discussion is provided to further facilitate a complete
understanding and appreciation of the present invention and its
ramifications.

1. Aging and Life Span

[0032]Aging in living organisms is more correctly termed senescence, and
is generally described as a cumulative, irreversible process resulting in
decreased function and increased risk of death. Aging of some kind
appears to affect all living organisms, from bacteria to humans
(Ackermann, Stearns et al., 2003; Stewart, Madden et al., 2005). How long
an individual lives--its life span--is characteristic of different
species (Finch, 1990) (e.g. Drosophila can live 100 days, humans can live
100 years). Within each species, life span is typically quite
variable--ven among individuals who are nearly genetically identical.
Comparisons of life spans between groups are therefore often reported as
mean and maximum life spans for the group or cohort.

[0033]The characteristic life spans of different species, and the variable
life spans of individuals within a species are determined by how their
unique genetic make-ups and environments make them more or less
susceptible to these mortality mechanisms. For example, in cold-blooded
(poikilothermic) animals like Drosophila, life span scales with
temperature across a broad range in both sexes. This suggests that there
is an irreversible, cumulative damage that leads to increased risk of
death of the organism. In addition, certain interventions such as dietary
restriction (DR) (Mair, Goymer et al., 2003; Partridge, Pletcher et al.,
2005) or a mild heat stress (Tatar, Khazaeli et al., 1997) can cause a
rapid and reversible shift in a population from a higher mortality rate
to a lower one, which demonstrates that there are more acute mechanisms
for regulating survival. Although the molecular nature of these mortality
mechanisms is unknown, oxidative stress, hydrolytic stress, and toxic
metabolite stress have each been implicated (Busuttil, Rubio et al.,
2003; Hekimi and Guarente, 2003; Gems and McElwee, 2005; Landis and
Tower, 2005; Wallace, 2005).

2. Evolutionary Theories of Aging

[0034]Like all things biological, aging and life span are shaped by genes
and evolution (Kirkwood and Austad, 2000). It is now common understanding
that deleterious mutations may be efficiently removed from the population
through natural selection. However, a mutation that causes a problem only
at late ages is not efficiently removed. For example, a human gene allele
that predisposes individuals to Alzheimer's or Parkinson's disease is not
efficiently removed from the population because by the time the disease
is manifested the gene has usually already been passed on to the next
generation (Finch and Sapolsky, 1999). The idea that such late-acting
mutations accumulate in the genome and create the aging phenotype
constitutes the "mutation accumulation" theory of aging.

[0035]The "antagonistic pleiotropy" model, on the other hand, suggests
that gene alleles with late-acting deleterious effects are maintained in
the population by active selection because these same gene alleles have
benefits during the developmental and/or reproductive stages.

[0036]Significant experimental evidence exists in support of both
mechanisms (Hughes and Reynolds, 2005). For example, the life span of
Drosophila can be increased in the laboratory by selecting populations
for late-life reproduction (Luckinbill, Arking et al., 1984; Rose, 1984;
Rauser, Tierney et al., 2006).

3. Asymmetric Gene Inheritance

[0037]Antagonistic pleiotropy, as described above, refers to gene alleles
that are beneficial at an early age and deleterious at a late age.
Another type of antagonistic pleiotropy is between the sexes (e.g. a gene
allele that benefits one sex of the species can be relatively deleterious
to the other sex) (Rice, 1992; Rice, 1998; Chippindale, Gibson et al.,
2001). This is possible because the sexes have different genotypes (e.g.,
X/X vs. X/Y), different environments (e.g., unique genital tract
microbial fauna) and different selective pressures (e.g., childbirth).
These sexually antagonistic genes are expected to be more likely to
contribute to the aging phenotype. That is, all other things being equal,
a gene allele optimized for function in both sexes is less likely to
cause a problem during aging than a gene allele that functions
sub-optimally in one or both sexes.

[0038]For example, the Y chromosome and the X chromosome are
asymmetrically inherited in both Drosophila and humans. In both species,
females are genotype X/X and males are X/Y (FIG. 1). These two genomes
may be differentially optimized for each of the sexes and have different
contributions to the aging phenotype.

[0039]Let's first consider the Y chromosome. Since the Y chromosome is
inherited only through the male, it means that genes on the Y chromosome
are optimized for function only in males. Evidence for the better fitness
of Y to the male sex is found in the fact that genes on the Y chromosome
are generally involved in male-specific functions such as spermatogenesis
and male sexual differentiation (Charlesworth and Charlesworth, 2005).

[0040]In contrast, the X chromosome exists in both the male and the
female. However, because females have two copies of the X chromosomes, it
follows that females have two copies of each gene on the X chromosome
whereas males only have one copy. This means that through evolution,
genes on the X chromosome spend more time under selection in females than
they do in males. The difference in evolutionary time exposure in the two
sexes suggests that there might be some skew in the distribution of genes
with sex-specific functions between the X chromosome and the autosomes.

[0041]Data from the genome projects appear to support this hypothesis. For
example, in Drosophila, the X chromosome is about half the size of the
2nd and 3rd chromosomes (the estimated number of genes on the X is about
2309 (˜16%), about 5688 (˜40%) on the 2nd, and about 6302 (or
44%) on the third, out of a total of about 14384. On the Drosophila X
chromosome, genes involved in oogenesis appear to be over-represented
whereas genes involved in spermatogenesis are under-represented, as might
be expected for a chromosome better optimized for the female (Table 1).

[0042]The same skew exists for genes showing femalebiased or male-biased
expression at the RNA level (Arbeitman, Furlong et al., 2002; Parisi,
Nuttall et al., 2003; Oliver and Parisi, 2004; Parisi, Nuttall et al.,
2004). In addition, the X chromosome was also found to be a hotspot for
sexually antagonistic fitness variation, i.e., naturally occurring X
chromosomes often contain gene alleles that benefit one sex more than the
other in terms of traits like male reproductive success and female
fecundity (Gibson, Chippindale et al., 2002).

4. Mitochondrial Asymmetric Inheritance

[0043]Because mitochondria are asymmetrically inherited (maternal
inheritance only), the force of natural selection acting on the
mitochondrial genome (mitogenome or M) and the mitogenome-nuclear genome
interactions is effective only in females (FIG. 1) (Rand, Clark et al.,
2001; Rand, 2005; Rand, Fry et al., 2006). In other words, the
mitochondrial genome is optimized for function in the female. The male is
inherently less fit because the highly beneficial mitochondrial genome is
not optimized for his genome. As set forth above, this asymmetric fitness
of the X genome will contribute to the observed aging phenotype, and
perhaps do so more in males than females. This is one possible
explanation for the observation that in many species, including
Drosophila and humans, males tend to have shorter life spans than
females.

[0044]At the genome level, it was found that genes involved in general
mitochondrial function were fairly evenly distributed (17% vs. 36% and
47%, respectively; Table 1). However it is interesting to note that genes
involved in programmed cell death are reduced in abundance on the X
chromosome, especially genes with anti-apoptotic function. Out of the 28
genes involved in programmed cell death found on the X chromosome (Table
2), pro-apoptotic functions outnumber anti-apoptotic functions 25 vs. 3.
The observation that (anti)apoptotic genes are preferentially located on
the autosomes (like spermatogenesis genes) suggests some degree of sexual
antagonism with regard to apoptosis regulation.

5. Apoptosis

[0045]Apoptosis is a form of active cellular suicide involving
characteristic morphological changes such as membrane blebbing. It
functions to remove cells that are otherwise unwanted, such as in the
developmental sculpting of human fingers and Drosophila gut, or
dangerous, such as virally-infected cells (Baehrecke, 2002; Baehrecke,
2003; Cashio, Lee et al., 2005; Yin and Thummel, 2005).

[0046]An evolutionarily-conserved set of cysteine proteases called the
caspases carry out most of the cellular self-destruction (Abraham and
Shaham, 2004). The caspases exist in a relatively inactive state in
virtually all cells of eukaryotes where they are regulated by a balance
of specific activators and inhibitors. The mitochondria regulate
apoptosis by releasing cytochrome C and other pro-apoptotic factors into
the cytosol in response to various "death signals" (Adams, 2003). These
signals include nuclear DNA damage, p53 activation and the balance of
Bcl-family member activities. The released cytochrome C binds to Apaf-1
protein which promotes assembly of a multiprotein complex called the
apoptosome and activates the initiator caspase-9 and a "caspase cascade"
(Adams and Cory, 2002; Arama, Bader et al., 2005).

[0047]Since apoptosis is essential to the development and function of both
male and female animals, both sexes must be capable of regulating a basic
cellular apoptotic machinery.

6. The Mitochondria and Apoptosis in Gametogenesis

[0048]Mitochondria play a central role in the differentiation of the
gametes in Drosophila and other species. Mitochondrial rRNA appears to be
an essential component of the germ plasm--the maternal, cytoplasmic
determinant of germ-cell fate (Amikura, Sato et al., 2005; Kobayashi,
Sato et al., 2005).

[0049]During Drosophila oogenesis, ribosomal RNAs encoded by the
mitochondria are transported out of the mitochondria into the cytosol of
the oocyte where they are required for formation of the morphologically
distinct germ plasm. The cells that inherit the germ plasm during the
development of (male or female) embryos give rise to the germ cells in
the adult. This is consistent with an ancient role for the mitochondria
in the evolution of sexual differentiation and germ-line/soma
distinctions.

[0050]The observation that mitochondrial genes are almost never inherited
through the male means one or both of two things (Nishimura, Yoshinari et
al., 2006): (i) There exists a male-specific mechanism for destruction of
functional mitochondrial genomes, or (ii) the mitochondrial genomes that
are delivered to the oocyte from sperm are non-viable in the oocyte or
embryo environment. In either event we can state that there is a
female-specific mechanism for mitochondrial inheritance, and males either
lack or for some other reason do not express this mechanism (FIG. 1).

[0051]The dramatic behavior of mitochondria in germ cells offers some
clues to the underlying molecular mechanisms. For example, oogenesis in
many species is characterized by a morphologically distinct aggregate of
mitochondria and other cytoplasmic material called the Balbiani body
(Kloc, Bilinski et al., 2004; Wilk, Bilinski et al., 2005). During
development, Drosophila oocyte is connected to its sister germ-line cyst
cells via cytoplasmic bridges, and the Balbiani body and a specialized
actin structure called the fusome mediate early movement of mitochondria
into the oocyte (Cox and Spradling, 2003). It may be that only this early
population of oocyte mitochondria is incorporated into the germ plasm to
be inherited by the next generation.

[0052]Later the cyst cells dump more mitochondria into the oocyte
cytoplasm prior to--or coincident with--undergoing a form of programmed
cell death (Buszczak and Cooley, 2000). Subsequently a significant number
of oocytes and eggs may be destroyed and reabsorbed in the Drosophila
ovary, in a process modulated by insulin-like signaling (Drummond-Barbosa
and Spradling, 2001; Flatt, Tu et al., 2005).

[0053]Germ-line cysts, mitochondrial transport mechanisms and multiple
apoptotic-like processes also appear to function in mammalian oogenesis
(Pepling and Spradling, 2001; Hussein, 2005). In adult mammals, most
female germ cells are destroyed prior to fertilization in an
apoptosis-like process called atresia. It has been hypothesized that
atresia is one mechanism to remove oocytes carrying mutant mitochondria
and thereby ensure the inheritance of functional mitochondria by the next
generation (Krakauer and Mira, 1999).

[0054]In summary, female inheritance of mitochondrial genomes appears to
be accomplished by active transport and concentration of (probably a
subset) of mitochondria into the oocyte, and perhaps the destruction of
cells containing unwanted mitochondria.

[0055]In the male germ line the mitochondria undergo a series of dramatic
transformations linked to the morphological development of the sperm, and
ultimately give rise to highly derivative structures containing only a
small fraction of the starting mitochondrial DNA (e.g., the mammalian
sperm midpiece and the insect sperm "nebenkern"). In Drosophila an
apoptosis like process has been found to be essential for normal sperm
development, in particular the spermatid individualization step in which
most of the cytoplasm and the majority of the mitochondria are eliminated
from the developing spermatids (Fabrizio, Hime et al., 1998; Arama,
Agapite et al., 2003; Arama, Bader et al., 2005; Cashio, Lee et al.,
2005).

[0056]As a further hint for the underlying molecular mechanism, ectopic
expression of the baculovirus caspase-inhibitor gene p35 in the testes,
or mutation of the Drosophila homologs of Cytochrome C, Apaf or Caspase-9
are found to cause severe defects in this apoptosis-like process. These
essential apoptosis events are attractive as a possible mechanism for
sperm-specific destruction of mitochondrial genomes. In the unicellular
alga C. reinhardii and the Japanese pet fish O. latipes, the fate of the
mitochondrial DNA has been examined in detail, and in each case what
little male mitochondrial DNA makes it to the egg is actively destroyed
just after fertilization (Nishimura, Yoshinari et al., 2006).

7. Evolution--the Benefit

[0057]The asymmetric inheritance of the M, X and Y chromosomes creates
abundant opportunities for antagonistic pleiotropy of gene function
between the sexes. This sets up a situation of balancing competition and
selection between the male and female which is thought to benefit both
because it promotes genetic diversity--sometimes called a Red Queen
situation (Nowak and Sigmund, 2004). One may envision this situation as a
driving force in eukaryotic evolution as follows: The male is inherently
less fit because he receives an M that is not optimized for his genome.
Since selection cannot act in the male to improve M gene function, it
acts to improve the fit of the male genome to the M. Because of this,
there is a strong selection force in the male to select for mutation of
the X so as to compensate for his lack of fitness--as opposed to the
autosomes, which they share equally. This leads to hypermutation of the X
in the male: Such X-linked mutations will be heterozygous in his
daughters and might benefit him and his grandsons. Since the male is
characterized as having suboptimal mitochondrial function, it is likely
that the hypermutation of the X in the male might proceed primarily
through oxidative mechanisms.

[0058]Strong selective pressure is predicted to act on the male to make
spermatogenesis and sperm success as dependent upon the mitochondria as
possible, e.g., motile sperm, regulated apoptosis and the elaborate
morphological changes. In this way the male "forces" the female to give
him as good M genes as possible. In other words, the males have created a
limit to the extent to which the female can make the mitochondria
sub-optimal for his genome, because if she makes it worse her eggs won't
get fertilized. This is the likely explanation for the fact that across
species males are found to produce large numbers of sperm, all of which
appear to be marginally functional. Natural selection in the male and
female will act on the X and on X-autosome interactions to create
ever-more distinct gametogenesis mechanisms--in order to "force" the
opposite sex into providing them with as good a set of genes as possible
(e.g. natural selection acts in the female to make oogenesis as dependent
upon the X and X-autosome interactions as possible to try to force males
into giving her as good (and un-mutated) X chromosomes as possible). This
dance between the male and female through time drives the evolution of
multicellularity and the separation of germ-line and soma. For example,
natural selection acts in the male to make spermatogenesis as dependent
upon the mitochondria as possible, including the elaboration of a
separate and disposable soma that houses and supports spermatogenesis and
is highly dependent on mitochondrial function.

[0059]The same general rules can be extended to mating choice for several
species. For example female Drosophila select males based primarily on
energetic (mitochondriadependent) traits--he chases her. In turn, males
are predicted to select females based primarily on potential for maternal
contribution (e.g., size of reproductive tissues) and X chromosome
genetic diversity (e.g., odor). Consistent with this idea, X chromosome
gene expression appears to be especially variable in human females
(Carrel and Willard, 2005).

[0060]The evolutionary considerations set forth above may be used to make
predictions as to what types of genes are more likely to exhibit
antagonistic pleiotropy and be involved in limiting the life span of
flies and mammals--specifically, genes controlling mitochondrial function
and sex-specific functions such as gametogenesis, sex-determination,
sex-specific differentiation, behavior and metabolism.

8. Life Span Quantitative Trait Loci (QTLs)

[0061]Quantitative trait loci (QTLs) are regions of the chromosome that
are associated with differences in a scalable phenotype such as bristle
number or life span. Life span QTLs can be identified based on the
general strategy of crossing a short-lived strain with a long-lived
strain, deriving sub-strains of varying life span, and correlating
specific chromosomal genetic markers with shorter or longer life span
across strains. This strategy works quite well in organisms such as
Drosophila, C. elegans and mouse and QTLs affecting life span have been
identified in several labs (Nuzhdin, Pasyukova et al., 1997; Leips and
Mackay, 2000; Vieira, Pasyukova et al., 2000; Jackson, Galecki et al.,
2002; Mackay, 2002; Ayyadevara, Ayyadevara et al., 2003; Valenzuela,
Forbes et al., 2004; Wang, Lazebny et al., 2004; Hsu, Li et al., 2005;
Nuzhdin, Khazaeli et al., 2005).

[0062]One of the most striking observations from these studies is the
degree of sex-specificity of the QTLs--many or most of the life span QTLs
identified in both Drosophila and mouse are sex-specific, and their
effects can be modified by mating (Reiwitch and Nuzhdin, 2002). This has
led to the conclusion that antagonistic pleiotropy of gene function
between sexes and developmental stages shapes life span (Vieira,
Pasyukova et al., 2000; Leips, Gilligan et al., 2005).

[0063]Although it is difficult to go from QTL to a specific gene, there
have been some successes (De Luca, Roshina et al., 2003; Miller, 2005).
For example the Drosophila gene DDC catalyzes the final step in the
synthesis of the neurotransmitters dopamine and serotonin and affects
both courtship behavior and life span.

9. Life Span Mutations and Transgenes

[0064]A number of single-gene mutations have been identified that can
increase Drosophila life span (Helfand and Rogina, 2003; Ford and Tower,
2006). Where tested, most of the mutations appear to affect both male and
female, although there is often a bias in effect for one sex or the other
(Burger and Promislow, 2004).

[0065]In one example, ubiquitous over-expression of the antioxidant enzyme
Cu/ZnSOD in Drosophila was found to increase life span in both male and
female flies (Sun and Tower, 1999). Cu/ZnSOD is found in the cytoplasm
and outer mitochondrial space in most eukaryotic cells (Landis and Tower,
2005). Using two independent huCu/ZnSOD transgenes, the preferential
over-expression of human Cu/ZnSOD (huCu/ZnSOD) in Drosophila
motorneurons, was also found to increase life span in males and females,
(Parkes, Elia et al., 1998). Interestingly, a recent analysis of one of
those huCu/ZnSOD transgenes in several long-lived genetic backgrounds
found life span extension to be primarily in females (Spencer, Howell et
al., 2003). This might indicate some sex bias in the mechanism of life
span extension by huCu/ZnSOD over-expression in Drosophila motorneurons,
or might represent a sex bias in the expression of that one particular
transgene insertion.

[0066]Another striking example of what could be sexually antagonistic gene
function is a seminal fluid protein (produced in the Drosophila male)
that may help his sperm compete against other male's sperm--yet at the
same time shortens the life span of the inseminated female (Wolfner,
2002). The fact that genes can be expressed in one sex but function in
the other sex, either through insemination or maternal contribution to
the embryo, provides ample opportunities for the evolution of sexually
antagonistic gene functions.

[0067]In yet another example, a conserved insulin-like signaling pathway
has been identified that negatively regulates life span in C. elegans,
Drosophila and mice (Bartke and Brown-Borg, 2004; Kenyon, 2005). In
Drosophila, inhibition of the insulin-like pathway or transgenic
over-expression of the target transcription factor dFOXO increases life
span preferentially in females (Clancy, Gems et al., 2001; Tatar,
Kopelman et al., 2001; Hwangbo, Gersham et al., 2004). This suggests that
in Drosophila, insulin-like signaling normally limits life span more in
females than in males.

[0068]One possible explanation for the negative-life span regulation of
this pathway may be because this pathway regulates reproduction and
metabolism and females invest more metabolic resources in reproduction
than do males.

[0069]Dietary restriction (DR) also increases life span to a greater
extent in Drosophila females than it does in males (Magwere, Chapman et
al., 2004). A mild stress applied early in life can sometimes increase
the life span of an animal, an effect called hormesis (Cypser and
Johnson, 2003). In Drosophila, mild heat and other hormetic stresses tend
to benefit males more than females (Vieira, Pasyukova et al., 2000;
Burger and Promislow, 2004).

[0070]There are also a small number of interventions and genes that have
been shown to increase life span in rodents (Miller, 2005). DR increases
both male and female life span, but may do so more in females (Masoro,
2005). Ames dwarf mouse, Snell dwarf mouse, and Little dwarf mouse
represent mutations in the insulin-like signaling and growth hormone
pathways and increase life span in both sexes, again with a preference
for females (Bartke, 2005). Strikingly, in the Ames dwarf mouse,
extension of life span correlates with an almost complete loss of gender
dimorphism in the gene expression patterns observed in the liver
(Amador-Noguez, Zimmerman et al., 2005). This was interpreted to suggest
that a reduction in costly physiological investments in reproduction
contributes to extended longevity.

[0072]Regulation of life span by the insulin-like pathway in the
hermaphrodite nematode C. elegans correlates with levels of oxidative
stress resistance (Larsen, 1993). Life span extension occurs in the adult
and is mediated by a set of genes including small heat shock proteins and
ones similar to the classic Phase II response involved in detoxification
and excretion of lipophilic metabolites (Walker, White et al., 2001; Lee,
Kennedy et al., 2003; Murphy, McCarroll et al., 2003; An, Vranas et al.,
2005; Gems and McElwee, 2005).

[0073]Interestingly, one of the major targets of reduced insulin-like
signaling is the mitochondrial antioxidant MnSOD (Honda and Honda, 1999)
--which has been shown to be sufficient to increase life span in adult
flies (Sun, Folk et al., 2002).

[0074]The ability to inhibit specific gene expression in C. elegans by
simple feeding of dsRNA has allowed for genome-wide screens for negative
regulators of life span, and the assessment of when during the life cycle
these genes function to inhibit life span. In addition to the
insulin-like signaling pathway, a major class of genes identified were
ones with mitochondrial functions (Dillin, Hsu et al., 2002; Lee, Lee et
al., 2003). The data suggest that a large number of mitochondrial genes,
and presumably the mitochondria itself, function during C. elegans
development to limit the life span of the subsequent adult. Taken
together, the data suggest that in C. elegans the mitochondria can
function during development to limit subsequent adult life span, and can
function in the adult to promote life span.

[0075]So far there is no indication that these life span effects involve
apoptotic-like mechanisms. Virtually all experiments were done in
hermaphrodites, so few male/female comparisons are available (McCulloch
and Gems, 2003).

[0076]In summary the genetic and transgenic studies clearly support a role
for mitochondria-related genes and functions in aging and life span
regulation across species, with hints of important sex-specific
differences. The predicted importance of other sex-specific genes and
pathways is indicated by the QTL studies, but remains to be confirmed by
the identification of specific genes with differing effects on male and
female life span. The trends that have emerged so far are that female
life span may be more limited by the insulin-like signaling pathway and
DR, while male life span may be more limited by (oxidative) stress.

10. Oxidative Stress and Apoptosis in Old Animals

[0077]A large body of data demonstrates a correlation between
mitochondrial misfunction, oxidative stress and aging across species
(Walter, Murasko et al., 1998; Hekimi and Guarente, 2003; Fridovich,
2004; Landis and Tower, 2005; Wallace, 2005). During aging, the
concentration of oxidatively-damaged macromolecules and abnormal
mitochondria are increased and the oxidative stress-response genes are
expressed in tissue-specific patterns. These observations appear to apply
generally to both males and females of Drosophila, rodents and humans. As
specific molecular markers for apoptosis become available, it has become
apparent that apoptosis is also occurring during aging in tissue-specific
patterns in Drosophila and mouse, however there has been little if any
comparison of male vs. female patterns (Kujoth, Hiona et al., 2005;
Zheng, Edelman et al., 2005).

11. Role of Apoptosis in Regulating Life Span

[0078]The observation of apoptotic events in old animals begs the question
of whether this process limits life span. Results of a genetic screen
support a role for apoptosis in Drosophila life span regulation (FIG. 2).

[0079]Previously, 10,000 male flies were generated where each fly had at
least one new insertion of an engineered P transposable element called
PdL (Landis, Bhole et al., 2003). PdL contains an outwardly-directed,
doxycycline(DOX)-regulated promoter at its 3' end, that can drive
over-expression of a gene downstream of the insertion site. The
longest-lived of the 10,000 male flies contained a single PdL insertion
causing over-expression of dIAP2--a known anti-apoptotic caspase
inhibitor with conserved function in humans (FIG. 2A). One hundred and
nine (109) strains were derived from the longest-lived flies and the
strains were re-tested for life span in cohorts of ˜400 male flies,
+/- DOX. dIAP2 over-expression in the presence of DOX yielded the
second-longest life span of all 109 lines and a +16% life span increase
relative to the control chromosome (FIG. 2B).

[0080]The dIAP2 mutation had not previously been pursued because there was
only a small difference between the +DOX and -DOX life spans (Landis,
Bhole et al., 2003). It now appears that this is due to the leaky nature
of the mutation and the potency of the gene product for life span effects
(Yishi Li and J. T., unpublished observations). A similar screen for life
span-extending mutations in Drosophila identified the dPOSH gene, which
may also be involved in apoptosis regulation (Aigaki, Seong et al.,
2002).

[0081]As demonstrated in Example 1, the apoptosis regulators p53 and
baculovirus p35 also regulate adult Drosophila life span. Finally,
Seroude and coworkers have recently found that inhibiting apoptosis in
Drosophila muscle tissue by over-expression of caspase inhibitors dIAP1
or baculovirus p35 increases both muscle function and life span (Personal
communication: Tissue-specific inhibition of apoptosis extends Drosophila
life span, J. Zheng, J. Yeung and L. Seroude, submitted). Taken together
the data suggest that, in Drosophila at least, apoptotic-like mechanisms
act in tissue- and developmental stage-specific ways to regulate life
span. However it should be noted that other studies indicate that p53 can
affect Drosophila life span via a mechanism other than apoptosis (Bauer,
Poon et al., 2005). Some preliminary data suggest intriguing sex-specific
differences in the way apoptotic regulators affect Drosophila life span
(Waskar et al, unpublished observations) and this should be a
particularly interesting area for future research.

12. A Binary Switch Model for Sex Determination, Apoptosis and Life Span

[0082]The inventor has devised a molecular model consistent with the data
and evolutionary theories using a binary switch metaphor--the on/off
status of a gene that regulates mitochondrial genome maintenance.

[0083]Because the mitochondrial genome is asymmetrically inherited, it
follows that there must exist some mechanism to ensure that the
mitochondrial genomes are inherited through the cytoplasm of the oocyte
and are (almost) never inherited through the sperm, as discussed above.
It is most likely that the asymmetric segregation is accomplished by a
mitochondrial inheritance system expressed in the oocyte that is not
expressed in the sperm, i.e., a mitochondrial inheritance system
downstream of the female germ-line sex determination pathway (FIG. 1).

[0084]One possible underlying molecular mechanism for this female-specific
mitochondrial inheritance is that only the mitochondrial genomes present
in the oocyte are licensed for replication--therefore any mitochondrial
genes coming in from the male would be diluted out, as appears to be the
case. Similarly, the mitochondria in the oocyte could be protected by a
female-specific anti-apoptotic mechanism. Mitochondria actively turn-over
in many cell types. Moreover, apoptosis is reported to be the default
state for the mitochondria (Jones, 2000; Brookes, 2005), which means that
in the absence of some anti-apoptotic signal, the mitochondria and its
genome will tend to self-destruct.

[0085]Regardless of the precise molecular nature of the female-specific
mitochondrial inheritance mechanism, it represents a mitochondrial
maintenance signal downstream of the female-sex determination pathway
(FIG. 1), and is most simply thought of as an anti-apoptotic signal sent
to the mitochondria.

[0086]In Drosophila, it has been found that both germ-line and somatic sex
determination as well as dosage compensation are controlled by the on/off
status of the Sxl (Sex lethal) gene (Birchler, Pal-Bhadra et al., 2003;
Graham, Penn et al., 2003; Bhadra, Bhadra et al., 2005, the entire
content of which are incorporated herein by reference). Sxl-on controls
female differentiation and therefore production of this theoretical
anti-apoptotic signal (FIG. 3).

[0087]How then are mitochondria maintained in the male soma and in sperm
precursor cells in the absence of this anti-apoptotic signal? There are
two possibilities. The first, and simplest, is maternal contribution of
the anti-apoptotic signal. The female would deposit in the egg enough of
the anti-apoptotic signal to support male development and
spermatogenesis, however the male is incapable of synthesizing the
signal. The second possibility is expression of the anti-apoptotic signal
(or some compensatory signal) in the male soma and sperm precursor
cells--but this requires another pathway for production of the signal and
it is not clear why the male would not utilize the same mechanism in the
sperm.

[0088]The first model seems most consistent with female control over
mitochondrial gene function. Perhaps the most intriguing prediction of
this model is that to a significant degree male mitochondrial function
and life span in flies (and humans) might be determined by the amount of
anti-apoptotic signal that he inherits maternally. A number of Drosophila
gene products are maternally supplied in quantities sufficient to produce
and function in the resulting adult animals--as evidenced by
maternally-rescued mutations (Table 3). These genes are good candidates
for encoding the anti-apoptotic signal and include Sxl itself, the Sxl
target gene daughterless and the anti-apoptotic gene Akt1.

[0090]In humans, there exists a gene that, like Drosophila Sxl, is on only
in females and that controls dosage compensation--the Xist gene (FIG. 4)
(Chow, Yen et al., 2005, the entire content of which is incorporated
herein by reference). Xist (or some other human female-specific gene)
could control an analogous female-specific anti-apoptotic pathway for
mitochondrial maintenance. The human female hormone estrogen has
anti-apoptotic properties and could be part of such a mechanism (Nilsen
and Brinton, 2004; Vina, Borras et al., 2005), and interestingly the
mitochondrial enzyme 17-β-estradiol dehydrogenase shows up in
several model organism studies of aging (Landis et al, submitted).

13. Why is Apoptosis the Default State for the Mitochondria?

[0091]A variety of genes, both nuclear and mitochondrial, co-exist in the
cell to their mutual benefit and thereby optimize their survival,
replication and transmission to the next generation. Genetic variation
and selection are the basis for evolution as we know it. For variation
and selection to occur, genes must give rise to new alleles and these
alleles must in turn segregate or otherwise re-assort--i.e., come apart
and re-unite in different combinations. From the point of view of any
given gene in the cell (Dawkins, 1976), it is beneficial for its partners
to vary i.e., leave and return, so that natural selection can act to
optimize its set of partners. The genes collaborating in the nucleus have
evolved an elegant and rather egalitarian mechanism to accomplish this
based on the spindle: independent assortment and recombination. But how
do the genes in the nucleus accomplish this segregation relative to the
genes in the mitochondria, and vice-versa? Natural selection has acted to
create a different mechanism by which the genes in the nucleus and the
genes in the mitochondria separate and re-unite over evolutionary
time--sex and asymmetric inheritance: In the sense of natural variation
and selection, the mitochondrial genes and nuclear genes are together in
the female and apart in the male.

[0092]Several observations demonstrate that the mitochondria and
mitochondrial genomes generally have a shorter functional half-life than
does the nucleus or cell. First, mitochondria are known to actively turn
over in many non-dividing cell types (Spees, Olson et al., 2006). Second,
as discussed above, the male germ line initially has cells with abundant
functional mitochondria, but ultimately gives rise to cells where the
mitochondria are absent or non-functional in terms of inheritance.
Clearly modern-day mitochondria are dependent upon the nucleus and
cellular milieu for growth and replication, but why should mitochondrial
apoptosis (self-destruction) be the default state?

[0093]From the point of view of the eukaryotic female cell this may be the
simplest way to control mitochondrial abundance and gene inheritance--to
engineer the mitochondria with an apoptotic mechanism and ration the
antidote. In other words, engineering the mitochondria with a shorter
half--life and rationing a survival/growth factor. From the point of view
of the mitochondria, this may be the simplest way to accomplish two
things: first to be maintained in the cell, and second, to be maintained
in the cell as an entity separate from the nucleus.

[0094]In general, the current state of affairs can be thought of in terms
of game theory as an evolutionarily stable strategy (ESS) for the
cooperation of the nucleus and the mitochondria (Nowak and Sigmund, 2004;
Burt and Trivers, 2006). As is typical of many game-theory strategies for
cooperation, one party (in this case the mitochondria) must sometimes
leave or defect. What does leaving or defecting amount to in a biological
context? Cellular apoptosis, organellar apoptosis, and asymmetric
segregation (a failure to be inherited) seem to be likely mechanisms.
With regard to inheritance and function, the mitochondria defects in the
male.

[0095]When the mitochondria invaded the eukaryotic cell (Gray, Burger et
al., 1999; Lang, Gray et al., 1999; Searcy, 2003; Timmis, Ayliffe et al.,
2004) it created competition between the nucleus and the mitochondria for
inheritance, as well as the potential opportunity for mutually beneficial
cooperation (FIG. 5). This sets up a situation similar to a "prisoner's
dilemma" in game theory (Nowak and Sigmund, 2004; Burt and Trivers,
2006): If the mitochondria always stays with the nucleus the nucleus will
absorb the mitochondria and it will cease to be a separate, multi-copy
entity. However, if the mitochondria sometimes leaves--i.e., is lost at
some finite rate, either by segregation or apoptosis--the nucleus must
actively maintain the mitochondria and this allows it to remain an
independent entity. In other words, shorter half-life and asymmetric
inheritance for the mitochondria represents an evolutionarily stable
strategy (ESS) for the co-operation of the nucleus and mitochondria. The
only way the mitochondria can be maintained as a separate entity from the
nucleus is to have a finite half-life, i.e., be lost at some rate by
segregation or apoptosis. This is the same thing as asymmetric
segregation: there are two states of the nucleus, with functional
mitochondria (e.g., egg) and without (e.g., sperm). This requires the
existence (or drives the evolution) of two states in the nucleus-one
state that prevents mitochondria loss and one that does not (e.g., Switch
Gene (SG)-on/off). In other words, the powerful selective advantage of
the mitochondria creates the sex determination gene and chromosome in the
nucleus: A successful and continued infection by the mitochondria would
require the existence of SG-on/off to establish and maintain the ESS. In
this model any asymmetrically inherited gene(s) with a large selective
advantage (like the mitochondrial genome) would define the female (more
fit), and the male (less fit).

[0096]It is possible to see X chromosome hypermutation in the male as an
attempt by the male to either activate or destroy the gene (SG) that
limits mitochondrial gene inheritance to the female, a process that might
in turn be hypothesized to drive the dynamic deterioration and evolution
of sex chromosomes (Charlesworth and Charlesworth, 2005; Graves, 2006).
Consistent with this idea, the gene at the top of the sex determination
pathway appears to mutate rapidly and change in identity often through
evolution (Graham, Penn et al., 2003) (Sxl in Drosophila melanogaster,
tra in Ceratitis capitata, and Xist in humans), and these genes are
predicted to exhibit antagonistic pleiotropy and function in regulating
life span. A change in the identity of the SG might be a handy mechanism
for speciation.

[0097]Interesting parallels can be seen between this model and what
happens when a largely detrimental genome such as the intracellular
parasite Wolbachia pipientis infects the Drosophila egg cytoplasm (Fry
and Rand, 2002; Starr and Cline, 2002; Fry, Palmer et al., 2004)
--successful infection can be dependent on the particular allele of Sxl.

14. Asymmetric Segregation of Genes as an Evolutionary Force

[0098]The general strategy of finite half-life creating asymmetric
segregation could be an ancient and important one in evolution. Consider
a primordial gene A that encodes a replicator molecule that replicates
gene A (FIG. 6). A and its product might be floating around free in the
primordial soup, or be surrounded by the membrane of a proto-cell
(Szathmary, 2000; Hogeweg and Takeuchi, 2003; Scheuring, Czaran et al.,
2003; Line, 2005). Another gene B could cooperate with and be linked to A
(either covalently or by inclusion in the same cell) but to be selected
for and maintained A+B must have greater fitness than A alone, such as by
encoding a better replicator. It is easy to see how this might work, but
if A and B are always linked together they are not separate genes. How
can A and B cooperate yet still exist and evolve as separate entities? As
mentioned above, for evolution to occur, a genes' partner(s) must somehow
vary as a function of time. One simple way for this to be accomplished is
if gene B has a shorter half-life than A (i.e., is lost at some finite
rate, i.e., ages). By definition, this creates two states for A: A by
itself and A+B.

[0099]In summary, a beneficial new gene with a shorter half-life by
definition creates asymmetric segregation, and asymmetric segregation by
definition creates increased complexity of the system. This ESS model
suggests that finite half-life (aging or senescence) is the consequence
of natural selection for increased complexity (evolution).

[0100]Is there any evidence that genes exist in such an ESS today? It is
interesting to note in this regard that the gene sequences with the
longest half-lives (i.e., most conserved through evolution) include many
polymerases, translation components, motor molecules and
transporters--perhaps representing the ancient master replicators. In
contrast, the most rapidly evolving genes include ones involved in
reproduction, especially male gametogenesis (Good and Nachman, 2005;
Nielsen, Bustamante et al., 2005; Richards, Liu et al., 2005). It also
seems possible that the DNA-end replication problem (Olovnikov, 1973;
Ohki, Tsurimoto et al., 2001) represents a strategy by which the
(ancient) DNA polymerase gene ensures that more distal genes on the
chromosome have a shorter half-life.

15. The Mitochondrial Apple

[0101]In Biblical history, the snake tempts Eve into eating an apple from
the forbidden tree of knowledge. Adam and Eve become aware of their
nakedness and in retribution God casts them out of the Garden of Eden
forever. When the proto-eukaryotic female ingested the highly beneficial
mitochondrial genome and maintained it through asymmetric inheritance,
she introduced an asymmetry in fitness between the sexes. The resultant
antagonistic pleiotropy of gene function between male and female helped
drive the evolution of multicellularity and ultimately self-awareness,
but came at a cost of aging phenotypes and limited life span.

[0102]Models for the co-evolution of sex and asymmetric inheritance are
not new, and include fascinating ones where sperm dynamics represent the
vestiges of the movement of the mitochondria's Rickettsia-like ancestor
from one cell to another (Fabrizio, Hime et al., 1998; Bazinet and
Rollins, 2003; Bazinet, 2004). Space considerations preclude discussion
of prokaryotic toxin/antitoxin systems (Gerdes, Christensen et al., 2005)
which seem eerily similar to the systems for mitochondrial inheritance
discussed here, or Honeybees--where expression of mitochondrial genes
distinguishes the long-lived Queen from the genetically identical
short-lived workers (Corona, Hughes et al., 2005).

[0103]Apoptotic cell death is implicated in many human aging-related
diseases, such as Alzheimer's disease and Parkinson's disease. However,
apoptosis has sometimes been discounted as a likely species-general
mechanism of aging based on the lack of detectable apoptotic cell death
in old C. elegans and the lack of effect of critical apoptosis genes such
as ced-3 caspase on C. elegans life span (Garigan, Hsu et al., 2002;
Herndon, Schmeissner et al., 2002). The current results suggest that
those conclusions should be reexamined in light of the fact that C.
elegans is a hermaphrodite, and predict that apoptosis might limit life
span in C. elegans males.

[0104]It has generally been assumed that mitochondria and oxidative stress
are consistently implicated in life span regulation (Fridovich, 2004)
implies that oxidative damage is inherently more toxic than the many
other damages and challenges cells suffer over time. However, in light of
the discovery of the present invention, the involvement of mitochondria
and oxidative stress in life span regulation may be explained by the
asymmetric inherited of mitochondrial genes which renders mitochondrial
functions more prone to antagonistic pleiotropy.

[0105]In view of the above, the model of the present invention provides a
basis for devising method for modulating aging and/or treating aging
related diseases such as Alzheimer's disease, Parkinson's disease,
cancer, Huntington's diseases, or any other aging related diseases known
in the art.

[0106]Additionally, anti-aging pharmaceutical products may also be
identified using methods of the present invention.

[0107]Last but not least, methods for in vitro evolution of genes or
products, diagnostic tools, and tools for performing aging related
research may also be advantageously devised.

[0108]Accordingly, in one aspect, embodiments of the present invention
provide a method for identifying a human gene sequence useful for
developing an anti-aging pharmaceutical product or a pharmaceutical
product for treating aging related disease, comprising obtaining Xist
gene sequences of a population of long-living individuals; and
correlating the Xist gene sequences to sex and age dependent
characteristics, wherein sequences having high correlation to sex and
old-age define template sequences for designing said pharmaceutical
products.

[0109]It is an unexpected discovery of the present invention that the Xist
gene plays a role in regulating the life-span of an individual.
Accordingly, an agent that is capable of activating the Xist gene in an
individual may be used as a therapeutic to prolong life or to treat aging
related diseases. By correlating variations in the gene sequence to a
population of long-living individuals, sequences or subsequences of the
gene that are correlated to long life span may be identified.

[0110]In a preferred embodiment, the population for performing such a
correlation analysis should consists of only individuals of age 80 and
above, more preferably 100 and above. Other characteristics may also be
selected for correlation analysis. Exemplary characteristics may include,
but not limited to disease history, behavioral characteristics, or any
other characteristics of interest.

[0112]Exemplary native Xist gene sequence that may be used for the
correlation analysis is as provided in SEQ ID: 3, or any other fragments
thereof, alternate splicing products thereof, or any combinations
thereof.

[0113]In another aspect, embodiments of the present invention provide a
method for manipulating the aging process or treating an aging related
disease, comprising: administering a pharmaceutically active composition
comprising a Xist RNA, alternate splice forms of the RNA, a fragment
thereof, or an analog thereof to a subject.a method in accordance with
the present invention for treating aging in a subject generally comprises
the steps of administering to a subject an agent capable of activating
the expression of Xist gene in the subject.

[0114]Exemplary means of administering the pharmaceutically active agent
may include, but not limited to direct inject, oral ingestion, or by way
of a biological delivery vector. Exemplary vectors may be any suitable
vector generally known in the art so long as it is capable of delivering
the Xist gene product to a host cell.

[0115]In yet another aspect, embodiments of the present invention provides
a method for treating aging or aging related diseases, comprising
administering to a subject an agent capable of activating the expression
of Xist gene in the subject.

[0116]Exemplary agents capable of activating the expression of the Xist
gene may include, but not limited to a nucleic acid, a polypeptide, a
protein, a nucleic acid mimetic, a small organic molecule, or a
combination thereof. Given a Xist gene sequence (as exemplified by SEQ
ID: 3), the design of an agent capable of activating the gene may be
achieved by molecular biology techniques commonly known in the art. In
one preferred embodiment, the agent is 5-azacytadine or an analog
thereof.

[0117]In yet another aspect, embodiments of the present invention provides
a method for in vivo evolution of genes to obtain genes having desired
properties, comprising: selecting a starting set of genes with high rates
sequence variation, wherein at least one gene is capable of cleaving
itself at a predetermined rate; replicating the genes via in vivo
replication to evolve the genes; and testing the properties of the genes
at predetermined intervals for the desired property.

[0118]To implement finite half-life of genes for in vivo evolution of
genes with desired properties, the staring gene would be engineered to
both be able to replicate itself with an significant rate of sequence
variation, and to also have short half-life by being able to cleave
itself at a finite rate. Alternatively the substance of which the gene is
composed would be designed to have a short half-life due to dissolving in
the aqueous media, or by rapid thermal denaturation. The gene may be
composed of RNA, DNA, protein, sugar/carbohydrate, lipid, or some
modification or combination of those compounds. One typical example is a
catalytic RNA that can both replicate itself and cleave RNA and that
contains its own recognition site for cleavage. Another example is a
protein that can replicate itself, alone or in combination with other
proteins, and that can also cleave protein and that contains its own
recognition site for cleavage.

[0119]In yet another aspect, embodiments of the present invention provides
a microarray useful for studying genetic regulation of aging, comprising:
a plurality of nucleic acid sequences wherein at least one sequence
comprises a Xist gene sequence, a Sxl gene sequence, or a mutant thereof.

[0120]General methods for manufacturing of microarrays are known in the
art. Microarrays according to embodiments of the present invention may
contain specific Xist gene sequences or Sxl sequences identified from
sex/age population analysis such as described in the methods above. The
microarrays may be useful both as a research tool and a clinical
diagnostic tool.

[0121]To assay the specific sequence and expression pattern of various
splice forms of Xist in individuals, sub-sections of Xist gene sequences
can be affixed to a solid support, such as a glass slide or other
material, in the form of DNA or oligonucleotide, to generate a
micro-array. DNA and/or RNA samples obtained from individual patients can
then be chemically labeled and hybridized to the micro-array to allow for
rapid screening and identification of the individuals Xist gene sequence
and expression pattern.

[0122]Patient samples might be from blood or serum or other tissues. This
should allow for rapid assessment of the individuals aging rate and
predisposition for specific aging-related diseases.

[0123]In yet another exemplary embodiment, there is provided A kit useful
for studying genetic regulation of aging, comprising: at least one vector
comprising a Xist gene sequence, a Sxl gene sequence, or a mutant
thereof. Similar to the microarrays, the kit may comprise specific Xist
sequences or Sxl sequences identified from sex/age population analysis.
Furthermore, kits according to embodiments of the present invention may
also be in the form of a library of vectors containing Xist gene sequence
or Sxl gene sequences.

[0124]To further illustrate the present invention, the following specific
examples are provided.

EXAMPLES

Example 1

Drosophila Melanogaster p53 Acts to Limit Life Span

[0125]The p53 gene encodes a transcription factor that regulates apoptosis
and metabolism and is mutated in the majority of human cancers.
Drosophila contains a single p53 gene with conserved structure, and
expression of a dominant-negative p53 isoform in nervous tissue has been
shown to extend fly life span. Here analysis of multiple Drosophila p53
mutant genotypes and conditional over-expression of wild-type and
dominant-mutant transgenes revealed that p53 limits the life span of both
male and female flies, but acts to do so preferentially during male
development and in female adults. In contrast, wild-type p53 function
favored survival of both male and female adults under stress conditions.
Strikingly, over-expression of p53 or baculovirus p35 during development
was preferentially toxic to males, but in females produced a sub-set of
flies with increased longevity. Taken together, the data demonstrate that
Drosophila p53 has developmental stage-specific and sex-specific effects
on adult life span indicative of sexually antagonistic pleiotropy, and
support a model in which sexual selection maintains the aging phenotype
in both Drosophila and human populations.

Experiments

[0126]To test how p53 might affect Drosophila life span, flies that had a
deletion of the endogenous p53 gene were examined (FIG. 7). Various
trans-heterozygous p53 wild-type and mutant allele combinations were
assayed for life span simultaneously to help control for genetic
background effects. Experiments were done using a dextrose-containing
food that yields long life spans (L cohort), as well as on a food rich in
yeast and sugar that yields shorter life spans (W cohort). In both
experiments null mutation (-/-) of the p53 gene increased male life span
by about 30% relative to wild-type (+/+) controls, while heterozygous
(+/-) male flies had a smaller but significant increase (FIG. 8). Life
span was also increased in female p53 null mutant (-/-) and heterozygous
(+/-) flies relative to wild-type controls (+/+), however the effect was
less dramatic on rich food. Taken together these data suggest that p53
acts at some point in the life cycle to limit the adult life span of both
male and female flies, with greater effects typically observed in males.
In contrast, p53 gene function was found to favor the survival of both
male and female adult flies subjected to conditions of oxidative stress
(100% oxygen atmosphere) (FIG. 8C) and ionizing radiation (FIG. 2D). This
is consistent with previous reports that wild type p53 gene function can
protect Drosophila tissues from ionizing radiation and UV toxicity during
development.

[0127]To control for possible maternal effects and X chromosome effects,
several life span assays were repeated with the crosses done in both
directions simultaneously, i.e., varying which strain serves as mother or
father for the cross. An increase in life span of p53 null mutant (-/-)
flies relative to wild-type (+/+) controls was obtained in female progeny
regardless of cross direction, thereby ruling out a primary effect of
maternal genotype. Strikingly the p53P mutation is a P element insertion
predicted to disrupt expression of only the A isoform of p53 (FIG. 7),
and was associated with increased life span only in females.

[0128]The p53 protein functions as a quatramer with various protein
domains mediating multimerization, DNA binding and transcriptional
transactivation. Mutant forms of p53 lacking function of a particular
domain can have powerful dose-dependent effects that can either promote
or antagonize the activity of wild-type p53 referred to here as dominant
mutations. Several Drosophila p53 dominant mutations (M) were examined
and found to have complex effects on adult life span, depending upon the
particular allele, whether or not a wild-type copy of p53 was present in
the background, as well as the food composition. Some of the variability
in life span across genotypes is expected to result from differences in
genetic background. However, when considered as a group the dominant
mutations tended to decrease life span in males, and to increase life
span in females (FIG. 8 A, B). Since the dominant mutations should
generally antagonize wild type p53 functions, these results are
consistent with the results obtained above suggesting that p53 acts in
during male development and in adult females to limit adult fly life
span.

[0129]To confirm the effects of p53 on fly life span, the conditional
transgenic system Geneswitch (Ford et al. 2007, the entire content of
which is incorporated herein by reference) was used to over-express both
wild-type and mutant forms of p53. Over-expression of wild-type p53 in
adult flies had a strong negative effect on life span in females, but not
males (FIG. 9A). In contrast, over-expression of dominant mutant p53 did
not have a negative effect, and in fact female life span tended to be
increased (FIG. 9 C, D). This is consistent with the previous report that
over-expression of the p53 dominant allele using a non-condition system
increases adult female life span. Similar effects on adult female life
span were obtained using the FLP-out conditional system to over-express
wild-type and dominant p53 transgenes. Taken together, the
over-expression data suggest that p53 acts in adult females to limit life
span, with less effect observed in adult males. One possible mechanism by
which p53 might act in adults to preferentially limit adult female life
span might be by stimulating insulin/IGF-1-like signaling (IIS), since
IIS appears to preferentially limit life span in females of Drosophila
and other species. Another possibility might be that p53 interacts with
the dietary restriction (DR) mechanism, since DR preferentially increases
female life span in Drosophila and other species. Consistent with an
interaction between p53 genotype and DR pathway, here the effect of
dominant p53 mutations was markedly different in females depending on
food composition.

[0130]Almost opposite effects on adult life span were observed when p53
transgenes were expressed during larval development. Over-expression of
wild-type p53 during development was toxic to males and limited their
subsequent adult life span, but had less effect on females (FIG. 9).
Strikingly, titration of wild-type p53 over-expression during female
development produced some adult females with unusually long life spans,
demonstrating that p53 can sometimes act during female development to
promote the life span of the subsequent adults. Taken together the data
suggest that during development p53 acts preferentially in males to limit
the survival of the subsequent adults.

[0131]One mechanism by which p53 might act during development to affect
subsequent adult life span is by regulating apoptotic pathways. As such
the caspase inhibitor baculovirus p35 was tested to determine if any
similar effects on life span might be observed. When expressed during
development the baculovirus p35 gene was found to be preferentially toxic
to males and to dramatically limit subsequent adult male life span,
similar to the phenotypes observed with wild-type p53 above (FIG. 9).
Moreover, over-expression of baculovirus p35 during female development
resulted in a bi-phasic survival curve and a sub-population of female
adults with increased life span, again analogous to the results obtained
above with p53. Therefore the over-expression phenotypes of p53 and the
caspase inhibitor baculovirus p35 during development are similar,
suggesting that it may be an apoptotic pathway through which these genes
act during development to affect subsequent adult longevity in a
sex-specific manner.

[0132]In these experiments the p53 gene was found to favor the survival of
both sexes under oxidative stress conditions, yet to act at different
developmental stages to limit the life span of both sexes under normal
conditions. The female-specific longevity effect of the P element
insertion in the B-variant suggests that this isoform may preferentially
limit life span in females. It will be of interest in the future to
determine if the A variant might correspondingly act more to limit male
life span, and to determine if these variants display any corresponding
sex-specificity in expression patterns. Taken together the data are
consistent with a sexual antagonistic pleiotropy model in which p53
functions are maintained by positive selection for survival benefit in
each sex, despite having negative effects at another stage of the life
cycle in the other sex.

Methods and Materials

[0133]Drosophila culture and life span assays were performed as previously
described (Ford et al., 2007). Drosophila culture media for the W cohort
life spans used an older recipe including molasses, while all other
experiments used a newer recipe containing dextrose (data not shown).

[0135]FLP-out heat stress protocol. Age-synchronized cohorts of females of
each genotype were collected and maintained at 25° C. in groups of
20 flies per vial. The females were subjected to heat shock at 37°
C. for 90 min for either one day or two consecutive days at age 5-6 days,
as indicated. The females were combined with 5 young male flies (to
stimulate egg production) and transferred to fresh fly food vials every
other day and maintained at 25° C.

Analysis

[0136]Maximum life span was estimated as the number of days until 90% of
the cohort had died. A non-parametric Welch t-test was used to compare
the mean life span data between the different groups of p53 deletion
genotypes (data not shown). Differences in the shape of survival
functions and patterns of mortality were analyzed using a statistical
framework described by Pletcher. Individual life spans of p53 deletion
genotypes were fitted to Gompertz model: with probability density
function

f ( x ) = λ exp { γ t - λ
γ [ exp ( γ t ) - 1 ] }

[0137]The parameter lambda represents demographic frailty or baseline
mortality and gamma represents the rate of change in mortality with age,
the demographic rate of aging. To compare parameters among genotypes,
variables based on the Gompertz model were estimated by maximum
likelihood. To compare survivals curves, a likelihood ratio test was
used: The likelihood L0 under the assumption of a common lifetime
distribution is compared to the likelihood L1 when two different
Gompertz curves are assured. Under the hypothesis of common lifetime
distribution -2 log L0/L1 is approximately
chi2-distributed with 2 degrees of freedom. Following this
procedure, most p53 deletion genotypes were found to differ from (w/+;
+/+) controls (data not shown).

[0138]Unpaired, two-sided t-tests were used to compare mean life spans
between control and treated fly cohorts. One- and two-factor ANOVAs and
Welch's t-tests were used to ascertain whether p53 expression
significantly affects life span and fecundity (data not shown).

[0139]For FLP-out experiments mean life spans for females were compared
between control and heat pulsed populations using a two-factor
fixed-effects ANOVA model with main effects of genotype and heat pulse.
FLP-out over-expression of wild-type p53 had a significant negative
effect on mean life span (data not shown). To address FLP-out strain
effects on estimated maximum life span, nonparametric 95% confidence
intervals (CI) were calculated for 90% life span for flies in the control
(noheat shock) cohorts for each strain. Wild type p53 had [70, 78] 95%
CI, AF51 had [78, 80], B440-[80, 82], 259H-[79, 80] and control CI was
found to be [74, 76]. Each dominant negative strain (AF51, B440, 259H)
exhibited CIs that do not overlap with CI of control 90% survival age.
The 90% survival age is significantly different in each dominant negative
strain from control (p-value<0.005) using a permutation test (data not
shown).

Example 2

Molecular Misreading in Drosophila melanogaster

[0140]Molecular Misreading (MM) is the inaccurate conversion of genomic
information into aberrant proteins. For example, when RNA polymerase II
transcribes a GAGAG motif, it sometimes synthesizes RNA with a two-base
deletion. If the deletion occurs in a coding region, translation can
result in production of misframed proteins. Certain misframed proteins
increase in abundance during mammalian aging, and misframed versions of
human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in
neurodegenerative disease tissue. Here cDNA clones encoding wild-type
hApp and hUbb as well as frame-shifted versions (hUbb+1 and
hApp+1) were expressed in transgenic Drosophila using the
doxycycline-regulated system. Misframed proteins were abundantly
produced, both from the transgenes and from endogenous Drosophila
ubiquitin-encoding genes, and their abundance increased during aging.
Over-expression of hUbb was toxic during fly development, yet favored
survival when expressed in adults, while hUbbb+1 did not have these
effects. The data suggest that MM is an evolutionarily conserved aspect
of gene expression and aging, with specific phenotypic consequences.

Results

1. Generation and Conditional Expression of Transgenic Constructs

[0141]To determine if MM could be studied in Drosophila, cDNA clones
encoding wild-type and frame-shifted versions of the human proteins, hUbb
and hApp, were expressed in Drosophila using the conditional
doxycycline(DOX)-regulated system ("Tet-on") (Bieschke et al. 1998; Ford
et al. 2007). In the DOX-regulated system, the control and experimental
animals have identical genetic backgrounds, and transgene expression is
induced in larvae or adults by feeding the drug DOX. In this way any
possible toxic effects of the RNAs or proteins could be avoided since
expression should occur only in the presence of DOX. Human cDNAs encoding
wild-type proteins, and cDNAs engineered with the appropriate
dinucleotide deletions adjacent to or within the GAGAG motif were cloned
downstream of the DOX-regulated promoter (FIG. 10). These sequences were
introduced into Drosophila using P element mediated transformation and
multiple independent transgenic strains were generated for each
construct. In all the experiments presented, the strains homozygous for
the transgenic target constructs were crossed to the rtTA(3)E2 driver
strain (or other driver strains, as indicated), to generate hybrid
progeny containing both constructs. In the rtTA construct the powerful,
tissue-general cytoplasmic actin (actin5C) promoter drives expression of
the artificial transcription factor rtTA. Upon DOX feeding the rtTA
protein undergoes a conformation change and binds to specific sequences
(called TetO) in the target construct, thereby activating transgene
expression in all tissues except for the germ-line. For controls, the
rtTA(3)E2 line was crossed to Oregon-R wild type flies to generate hybrid
progeny containing only the rtTA(3)E2 driver construct and no target
construct, and for simplicity these controls are referred to hereafter as
"Or-R control" flies. Conditional (DOXdependent) expression of the
transgenes at the level of RNA transcripts was confirmed by Northern blot
(FIG. 11A, B).

[0142]The human ubiquitin-B gene encodes three direct repeats of ubiquitin
protein that is subsequently processed into mature monomers. The GAGAG
hotspot for MM is located at the 3' end of each repeat, such that MM
causes an almost full-length ubiquitin moiety to be fused with part the
next repeat in the +1 frame, thereby creating a altered ubiquitin species
with a C-terminal extension of the protein, called Ubb+1 (FIG. 10B).
The hUbbWT construct contains a single repeat designed to encode a
wild-type hUbb monomer. The hUbb+i construct contains two hUbb
repeats, with the appropriate dinucleotide deletion engineered at the
GAGAG hotspot at the end of the first repeat, thereby constitutively
encoding hUbb+1. The endogenous Drosophila Ubb-encoding genes
include a polyubiquitin (Lee et al. 1988) and fusions of Ubb to other
coding sequences that are conserved in mammals (Barrio et al. 1994).

2. Western Analysis of HUbbWT Expression

[0143]Western blot analysis with a specific antibody was used to assay for
expression of the hUbbWT protein in flies. The human and Drosophila Ubb
proteins are identical in amino acid sequence, so it was expected that
antibody raised against hUbb would cross-react with endogenous Drosophila
protein. Consistent with this expectation, the hUbb antibody recognized a
series of protein bands in Or-R control fly extracts, although notably no
band was detected at the ˜8.5 Kd size calculated for monomeric Ubb
(FIG. 11D, and additional data not shown). The lack of a detectable Ubb
monomer species is likely due to its rapid ligation to other proteins. A
scarce and limiting pool of free Ubb has previously been suggested to
explain the low abundance of Ubb monomers relative to multimers in
mammalian cell culture studies (Dantuma et al. 2006). The antibody
specific for Ubb recognized a series of high-MW proteins in the fly
extracts, several of which are indicated by a bracket (FIG. 10D). These
species are interpreted to represent endogenous Drosophila Ubb ligated to
various proteins in the cell. Importantly the abundance of these protein
species was not altered by DOX treatment in the Or-R control flies,
indicating that DOX itself does not have a detectable effect on ubiquitin
expression. A similar pattern of high-molecular-weight species were also
present in the extracts of transgenic flies where hUbb was being
expressed, and notably the abundance of these species was induced by DOX
in each of the three independent transgenic lines tested. These results
are consistent with DOX-dependent expression of hUbb from the transgenes
that is then rapidly ligated to fly cellular proteins.

3. Western Analysis of Ubb Molecular Misreading

[0144]To determine if expression of the misframed (+1) version of the hUbb
protein could be detected, antibody specific for hUbb+1 was used in
Western blot assays. This antibody had been previously characterized and
shown to be highly specific for hUbb+1 (van Leeuwen et al. 1998). As
expected this hUbb+1 antibody strongly recognized purified
His-tagged hUbb+1 protein purified from E. coli cells (FIG. 12A).
Strikingly, the Ubb+1 antibody also recognized a complex pattern of
bands in extracts of Or-R control flies that became more abundant with
age, including large amounts of high-MW material, as well as several
small species migrating at an apparent MW of <20 Kd (FIG. 12A). These
species are interpreted to represent Ubb+1 protein produced from the
endogenous Drosophila Ubb-encoding genes for two reasons: (i) the almost
perfect conservation of ubiquitin gene sequences between the fly and
human, including the GAGAG hotspot for MM, means that the endogenous fly
gene encodes the same Ubb+1 protein as does the human gene, (ii) a
similar pattern of DOX-inducible species was produced by both the
hUbb+1 and hUbbWT transgenes (FIG. 12B-F). The hUbb+1 transgene
produced a series of bands that cross-reacted with the hUbb+1
antibody, both small MW species as well as higher MW species. This
pattern of proteins was highly similar to that observed in the Or-R
control flies, and also appeared to include several additional species.
The calculated size for the Ubb+1 monomer is ˜11 Kd, and this
may correspond to one of the DOX-inducible species migrating at an
apparent MW of <20 Kd (indicated with black arrowheads in FIG. 12;
estimation of sizes not shown). Ubb+1 is itself known to be a target
for (poly)ubiquitination by wild-type Ubb (monomeric MW ˜8.5 Kd),
and notably a DOX-inducible species was present at the MW predicted for
Ubb+1 ligated to one Ubb moiety (˜19.5 Kd, indicated by an
open arrowhead), as well Ubb+1 ligated to two Ubb proteins
(˜28 Kd, indicated by asterisk) and Ubb+1 ligated to three Ubb
proteins (˜37 Kd, indicated by double asterisk) (estimation of
apparent MW not shown).

[0145]Strikingly, the hUbbWT transgenic strains produced a similar series
of bands whose abundance was induced by DOX and that cross-reacted with
the hUbb+1 antibody (FIG. 12B), consistent with MM events. These
included apparently the same small MW species described above, as well as
a similar series of higher MW species. The abundance of DOX-inducible
proteins cross-reacting with the Ubb+1 antibody was observed to increase
during aging of flies expressing both the hUbbWT and hUbb+1
transgenes (FIG. 12B-F). For example the ˜19.5 Kd species was more
readily detected in old fly extracts (open arrowhead). Taken together the
data suggest that Ubb+1 is abundantly produced in transgenic flies
from the hUbbWT transgene, consistent with MM, and moreover that the
abundance of these misframed hUbb protein species increases with age of
the flies. Notably for hUbbWT these MM events cannot be occurring at the
GAGAG hotspot (position 219 of the ORF), as it is located downstream of
the relevant epitope in this construct (indicated with red asterisk)
(FIG. 1B).

4. Western Analysis of hApp Expression and Molecular Misreading

[0146]Expression of hAppWT was assayed using a specific antibody (Upstate
Cat. #07-667), and no DOX-inducible species could be detected at the
calculated size of ˜79 Kd, or at other sizes (FIG. 11E), suggesting
that the hAppWT protein is not being expressed at a detectable level
and/or is not stable. Other studies have reported that hAppWT could be
expressed in adult flies and detected by Western blot at an apparent MW
of ˜110 Kd (Luo et al. 1992; Greeve et al. 2004). One possibility
is that hAppWT is being expressed at low levels in the experiments
presented here, but is being obscured by a background band such as that
running at ˜100 Kd (FIG. 11E; indicated with asterisk). However DOX
inducible expression of AppWT was also not detected using mouse
monoclonal antibody 22c11, which yielded a different pattern of
background bands (data not shown). We conclude that AppWT is either not
being expressed at a detectable level from this construct in adult male
flies, or that the protein is unstable. These hAppWT constructs are
indeed being expressed in a DOX-dependent manner at the RNA level, as
confirmed by Northern blots (FIG. 11B), and as indicated by the fact that
they give rise to hApp+1 via apparent MM events, as described next.

[0147]To determine if the misframed version of hApp could be detected in
flies, Western blots were performed using antibody specific for
hApp+1. The hApp+1 antibody readily detected His-tagged
hApp+1 protein purified from E. coli cells, as well as highly
abundant protein produced in flies transgenic for the hApp+1
transgenic construct at the same size, consistent with efficient
expression of hApp+1 in adult flies (FIG. 13A; indicated by black
arrowhead). Notably, both the His-tagged hApp+1 and the hApp+1
produced in transgenic flies ran in the gel at a position equivalent to
an apparent MW of ˜58 Kd, which is the reported mobility for
hApp+1 under these conditions (Hol et al. 2003). This is despite the
fact that the calculated MW for the 348 amino acid residue hApp+1
protein is ˜39 Kd. This unusual retarded mobility in SDS-PAGE gels
observed for hApp+1 (as well as hApp) has been observed in several
previous studies (Weidemann et al. 1989; Hol et al. 2003), and is
attributed to the acidic region of the protein between positions 230-260
that contains many glutamate and aspartate residues. In transgenic flies
expressing hAppWT transgene, a DOX-inducible band at the same apparent MW
of ˜58 KD was detected, consistent with MM of the hApp transgene
(FIG. 13C, D). It is also interesting to note that there were several
species in the Or-R control fly extracts that cross-reacted with
hApp+1 antibody, including one of a similar size as hApp+1
(indicated by an asterisk), and that these species became more apparent
with age (FIG. 13B). Despite this background, the fact that the
apparently ˜58 Kd species was produced in a DOX-inducible manner in
two independent hAppWT transgenic strains, but not in the controls,
suggests that MM is indeed occurring, and moreover that this hApp+1
protein is more readily detected in old flies.

5. Phenotypic Consequences of Expression of HUbbWT and hUbb+1

[0148]It was next asked if expression of wild-type and +1 versions of hUbb
transgenes would have phenotypic consequences for the flies.
Over-expression of the highly-expressed hUbbWT(70) transgene during
larval development was found to be toxic to flies at the pupal stage,
especially in males (FIG. 14), however no significant lethality was
associated with the less strongly expressing line hUbbWT(80). To confirm
that high-level expression was toxic, a strain was constructed containing
two copies of the hUbbWT(118) transgene along with the rtTA(3)E3
ubiquitous driver, and this combination resulted in pupal lethality that
was DOX-dependent and completely penetrant. The lethality caused by
hUbbWT over-expression was associated with a dramatic disruption of
normal pupae structures and large melanotic inclusions indicative of
extensive cell death (FIG. 14B). In contrast there was no evidence of
pupal lethality when the hUbb+1 transgenes were expressed during
development, using a variety of drivers. A different result was obtained
when the same transgenes were expressed in adult flies. In adults
hUbb+1 was found to have neutral or slightly negative effects on
survival, particularly in male flies (FIG. 14C, D). In contrast, hUbbWT
did not have these negative effects and instead was associated with
slightly increased life span (FIG. 14E, F). Notably the DOX-dependent
life span increase was greater in males, and was greater in the more
highly-expressed of the two lines, UbbWT(2)70. Moreover, in the
UbbWT(2)70 line a particularly long lifespan was observed even in the
absence of DOX, perhaps due to some leaky expression of the transgene.
Interestingly, in adult flies over-expression of hUbb was found to cause
increased expression of ribosomal protein 49 gene (Rp49) (FIG. 11).

6. A Molecular Misreading GFP Reporter Construct

[0149]The conditional DOX-regulated system was also used to express the
fluorescent protein GFP (the "Control-GFP" reporter), as well as a
reporter construct in which the GFP protein is frame-shifted to encode a
non-functional protein (the "MM-GFP" reporter) (FIG. 15). The MM-GFP
reporter contains 3 copies of a GAGAGA hotspot motif such that any MM
events should cause production of functional GFP. The Control-GFP
construct yielded abundant DOX-dependent expression of GFP throughout the
somatic tissues of the fly, as expected (FIG. 15B). Little or no
expression of GFP could be detected with the MM-GFP reporter in young
flies or during normal aging (data not shown). However, one possibility
is that the expression level was present but simply too low to detect. To
address this possibility strains are being generated with multiple copies
of the reporter in hopes of increasing the signal. Significant expression
of the single-copy MM-GFP reporters could be observed in leg muscle,
flight muscle and other tissues when adult flies were challenged with
ionizing radiation or 100% oxygen atmosphere (FIG. 15C). These are strong
oxidative stresses that are known to produce acute phenotypes with some
similarities to aging. Notably the expression often occurred in isolated
patches of tissue, which is reminiscent of the cell-by-cell accumulation
of misframed proteins previously observed in the Brattleboro rat
magnocellular neurons and human AD disease tissue (de Pril et al. 2006).
The data are consistent with MM events, and suggest the reporter may be
useful for studying MM in Drosophila longitudinal assays in the future.

Discussion

[0150]In these studies wild-type and misframed versions of Ubb and hApp
proteins were identified based on their apparent MW in SDS-page gels,
co-migration with proteins purified from E. coli, DOX-inducible
expression from transgenic constructs, and cross-reactivity with specific
antibodies. The Western blot analyses suggested that wild-type and
misframed versions of hUbb and misframed hApp proteins were successfully
expressed from the transgenes designed to encode these proteins. For both
the hUbbWT and hAppWT transgenes there was ample evidence of MM, as these
constructs produced DOX-dependent proteins that were recognized by
hUbb+1 and hApp+1 antibodies, respectively. Importantly these
hUbb+1 and hApp+1 species were more readily detected in
extracts from old flies, supporting the connection between MM and aging.

[0151]It was striking that the hUbb+1 antibody recognized a series of
abundant protein species in control flies. The fact that several of these
species appeared to co-migrate with DOX-inducible bands produced by the
hUbb+1 transgene (and hUbbWT transgene) supported their
identification as containing bona fide Ubb+1 protein. This suggests
that the endogenous Drosophila Ubb-encoding gene(s) are undergoing MM and
producing abundant Ubb+1 protein of various sizes, likely involving
cross-linking to other cellular proteins such as Ubb, and moreover that
these species become more abundant during aging. Finally, the production
of functional GFP protein from the misframed reporter construct suggests
that MM events take place in the tissues of flies challenged with
oxidative stress.

[0152]The ability of the hUbbWT transgene to yield expression of
DOX-inducible species that cross react with Ubb+1 antibody is
consistent with abundant MM, however these events cannot be occurring at
the GAGAG hotspot as it is located only downstream of the relevant
epitope in this construct (FIG. 10B). This suggests that one or more
other DNA sequence elements located in the 5' end of the wild-type human
ubiquitin gene are leading to MM, at least under the conditions assayed
here. The nature of these MM events is not clear at this time, as the
largest ORF containing the (+1) epitope in the hUbbWT construct does not
contain an ATG start codon, and would encode a protein of only 45 amino
acid residues (˜5 Kd). One interesting possibility is that the
DOX-inducible expression of the transgenes is affecting the expression
and MM of the endogenous genes and/or the stability and cross-linking of
endogenous Ubb proteins.

[0153]The faint pattern of endogenous Drosophila species cross-reacting
with the hApp+1 antibody most likely represents non-specific,
cross-reacting proteins, however it is not clear at this time why such
cross reactivity is more apparent in old fly extracts. The Drosophila
genome contains at least one gene related to hApp, the Appl gene, however
it is not obvious how it could encode a cross-reacting epitope or an
appropriately sized protein based on its known sequence (Luo et al.
1992). Analysis of fly strains mutant for Appl will be required to test
the intriguing possibility that Appl encodes proteins that cross-react
with antibodies directed against hApp and hApp+1.

[0154]One line of evidence in support of a phenotypic consequence for MM
is the effect of the over-expressed genes. While high-level expression of
hUbbWT was toxic to developing pupae, over-expression of hUbb+1 was
not, consistent with different functions for the two proteins. Moreover,
hUbbWT appeared to have benefits for survival of adult male flies, while
hUbb+1 had no benefit, or was slightly toxic. The ability of hUbbWT
over-expression to induce ribosomal protein 49 (Rp49) gene expression and
favor survival in adult male Drosophila is interesting in light of recent
reports that decreased translation can favor longevity in Drosophila
(Kapahi et al. 2004), C. elegans (Hansen et al. 2007) and yeast
(Chiocchetti et al. 2007). To what extent endogenous Ubb+1 may
function in normal cell physiology will be an interesting area for future
study.

[0155]The association of misframed proteins with AD and other disease
states and the ability of hUbb+1 to inhibit proteosome activity in
cultured cells in a dose-dependent manner is consistent with the idea
that accumulation of misframed proteins may be detrimental to the aging
animal. It will be important to determine if the increased abundance of
misframed proteins in old flies is due to increased rates of MM,
decreased clearance of the abnormal RNA species by NMD, decreased
turnover of the misframed proteins, or some combination of these
processes. Consistent with a toxic effect of accumulated protein damage
during aging, old flies are more sensitive to proteosome inhibitors
(Vernace et al. 2007), and over-expression of certain enzymes implicated
in protein repair such as protein carboxyl methyltransferase (Chavous et
al. 2001) and methionine sulfoxide reductase A (Ruan et al. 2002) are
reported to increase fly life span under appropriate conditions.

[0156]The fact that misframed proteins can have toxic effects and appear
to increase in abundance during aging in mammals and in flies is
consistent with an error catastrophe model, however other explanations
exist. For example the apparently abundant expression of Ubb+1 in
young, wild-type flies may indicate a normal physiological function.
Epigenetic regulation of gene expression and phenotypes is increasingly
apparent across species (Goldberg et al. 2007). Bistable switches are
common and appear to allow phenotypic plasticity on various timescales
(Rando and Verstrepen 2007). Interestingly repeated DNA sequence motifs
are commonly associated with such epigenetic mechanisms. Stress response
genes, particularly oxidative stress response genes such as hsps, are
induced during normal aging of flies as well as in human aging-related
disease states like AD (Landis et al. 2004; de Pril et al. 2006). The
genes encoding ubiquitin are induced in response to stress in flies
(Niedzwiecki and Fleming 1993) and mammals (Grillari et al. 2006), and
perhaps MM and the conserved GAGAG hotspot motifs represent an
evolutionarily conserved epigenetic mechanism by which ubiquitin genes
encode alternate proteins with alternate functions expressed in response
to certain kinds of stress. For example altered chromatin structure,
altered RNA polymerase structure, or low nucleotide concentrations might
each be predicted to increase rates of MM. The increased abundance of MM
in old flies might therefore represent a compensatory stress response
with a benefit for continued function of cells or the animal. Consistent
with this idea, in mammalian cells the expression of hUbb+1 caused
induction of hsp70 and increased resistance to oxidative stress (Hope et
al. 2003).

[0157]Alternatively, even if MM might serve some conserved beneficial role
earlier in the life cycle, such as in response to stress, its chronic
activation during aging might be counterproductive. The ability to
observe MM in the fly should facilitate the further study of this
intriguing phenomenon, including its possible relevance to human
aging-related diseases.

Methods and Material

[0158]Drosophila strains. All Drosophila melanogaster strains are as
described (Lindsley and Zimm 1992; Bieschke et al. 1998; Landis et al.
2001, the entire contents of which are incorporated herein by reference).

[0159]Plasmid construction. Transgenic constructs were generated by PCR
amplification of inserts with a PstI site engineered at the 5' end and an
EcoRI site at the 3' end, and these fragments were cloned into the unique
PstI and EcoRI sites of USC1.0 vector, as previously described (Allikian
et al. 2002). All construct sequences were confirmed by sequencing. For
hUbbWT construct, PCR products (UBBwt-1, UBBwt-2) were obtained using a
pcDNA3 vector containing the UBBwt cDNA as a template. UBBwt-1 was
generated using primers Uwt-1F (5' GGCTGCAGGAATTCGATATCAAGCT 3') and
Uwt-1R (5' TTTATTAAGGCACAGTCGAGGCTGATCAGCGA 3'). UBBwt-2 was generated
using primers Uwt-2F (5' TGCAGGCTGCAGGAATTCGATATCAAGCT 3') and Uwt-2R (5'
AATTTTTATTAAGGCACAGTCGAGGCTGATCAGCGA 3'). Both products were generated
using pfu DNA polymerase (Stratagene). Products UBBwt1 and UBBwt-2
were boiled for 10 min at 95° C. and cooled to room temperature to
generate a reannealed UBBwt gene with a PstI site engineered at the 5'
end and an EcoRI site at the 3' end. This fragment was cloned into the
PstI and EcoRI sites of USC1.0 (Allikian et al. 2002) to generate the
construct USC1.0-UBBwt. The following constructs, USC1.0-UBB+1,
USC1.0-APPwt, and USC1.0-App+1 were generated by using the procedure
above. UBB+1-1 was generated using primers U+1-1F (5'
GATCCATGCAGATCTTCGTGAAAAC 3') and U+1-1R (5'
TTTATTCCAGTGTGATGGATATCTGCAGAAT 3'). UBB+1-2 was generated using primers
U+1-2F (5' TGCAGATCCATGCAGATCTTCGTGAAAAC 3') and U+1-2R (5'
AATTTTTATTCCAGTGTGATGGATATCTGCAGAAT 3'). APPwt-1 was generated using
primers Awt-1F (5' GTGCTGGAATTCTGCAGATATCCAT 3') and Awt-1R (5'
TTTATTCGAGGTCGACGGTATCGATTCTTAA 3'). Appwt-2 was generated using primers
Awt-2F (5'TGCAGTGCTGGAATTCTGCAGATATCCAT 3') and Awt-2R (5'
AATTTTTATTCGAGGTCGACGGTATCGATTCTTAA 3'). App+1-1 was generated using
primers A+1-1F (5' TAGAACTAGTGGATCCCCCGGGAGA 3') and A+1-1R (5'
TTTATTCTCGTTGGCTGCTTCCTGTTCCAA 3'). App+1-2 was generated using primers
A+1-2F (5'TGCATAGAACTAGTGGATCCCCCGGGAGA 3') and A+1-2R (5'
AATTTTTATTCTCGTTGGCTGCTTCCTGTTCCAA 3'). Molecular misreading reporter
constructs. PCR products (GFP-1, GFP-2) were obtained using pGreen
Pelican plasmid containing the eGFP gene as a template. GFP-1 is
generated using primers PG1F (5'GTGAGCAAGGGCGAGGAGCT 3') and PG1R
(5'TTACTTGTACAGCTCGTCCA 3'). GFP-2 was generated using primers PG2F
containing 5'Sac I overhang (5' AGCTC GTGAGCAAGGGCGAG GAGCT 3') and PG2R
containing 3'EcoRI overhang (5'AATTTTACTTGTACAGCTCGTCCA 3'). Both
products were generated using pfu DNA polymerase (Stratagene). Products
GFP-1 and GFP-2 are then combined and boiled for 10 min at 95° C.
and cooled to room temperature to generate eGFP, a reannealed GFP gene
with a Sac I site engineered at the 5' end and an EcoRI site at the 3'
end. This fragment is then ligated to MM3X, which is a reannealed
synthetic oligos of MMF (5'ATGGAGAGAGAGAGAGAGAGATC GAGCTC 3') and MMR
(5'CGATCTCTCTCTCTCTCTCTCCATTGCA 3'). MM3X when annealed contains a 5'
Pst-I site and a 3' Sac I site which is complimentary to the 5'eGFP. MM3X
contains three copies of the GAGAGA molecular misreading hotspot. MM3X
was ligated to eGFP at 4° overnight and then USC 1.0 was added and
ligated at room temperature for 4 hrs to generate the construct
USC1.0-MMGFP. T 4 DNA ligase (Promega) is used in all the ligation
reactions. A control construct USC1.0-MMCGFP was generated by first
ligating eGFP gene to MM3XC, a reannealed synthetic oligos of MMFC
(5'ATGGAGAGAGAGAGAGAGAGAGAGCTC 3') and MMRC (5' CTCTCTCTCT
CTCTCTCTCCATTGCA 3'). The ligated product was then cloned into PstI and
EcoRI sites of USC1.0. P element mediated transformation. Four
independent germ-line transformants of the USC1.0-UBBwt construct
(hUbbwWT-8, -118, -80 and -70) were generated using standard methods
(Rubin and Spradling 1982), using the y-ac-w injection strain (Patton et
al. 1992). All four lines integrated onto the 2nd Chromosome. Six
independent germ-line transformants were generated for the
USC1.0-hUbb+1 construct. UBB+1-4, -1, and -11 integrated onto the
2nd chromosome while hUbb+1-6, -30, and -19 integrated onto the 3rd
chromosome. Southern analysis indicated the presence of single inserts
for all the lines. Four independent germ-line transformants were
generated for the USC1.0hAppWTconstruct (hAppWT-16, -24, -1, and -20).
hAppWT-16, -1 and -20 integrated onto the 2nd chromosome while hAppWT-24
integrated onto the 3rd chromosome. Four independent germ-line
transformants were generated for the USC1.0-hApp+1 construct
(hApp+1-7, -24, -16 and -30). hApp+1-16, and -30 integrated
onto the 2nd chromosome while hApp+1-7 and -24 integrated onto the
3rd chromosome. Six independent germ-line transformants were generated
for the USC1.0-MM-ATG-GFP construct (abbreviated "MM-GFP") (MM-ATG 58A,
46A, 34A, 8A, 46B, and 8B). MM-ATG 58A, 46A, and 34A integrated onto the
3rd chromosome and 46B and 8B integrated onto the 2nd chromosome. Two
independent germ-line transformants were generated for USC1.0MMC-ATG-GFP
construct ("Control-GFP") (MMC-ATG-GFP-16, -8). Drosophila culture and
life span assays. Drosophila were cultured on standard agar/dextrose/corn
meal/yeast media (Ashburner 1989). Where indicated, flies were cultured
on food supplemented to a final concentration of 640 μg/ml DOX for the
experimental group. Each of the hUbbWT(70,80), and hUbb+1(1,11)
transgenic strains, and Oregon R wild-type flies (provided by Bloomington
Drosophila stock center) was crossed to the "TO-daughterless" driver
line, which contains the daughterless-GAL4 driver and the "901" bridge
construct where a UAS-promoter drives expression of rtTAM2alt (Stebbins
et al. 2001; Ford et al. 2007). Crosses were performed at 25° C.
in urine specimen bottles. Prior to eclosion of the majority of pupae,
bottles were cleared of adult parents and newly eclosed flies were
allowed to emerge over the next 48 hours. Males and females each
containing both the target transgene and the driver constructs were
scored and collected. At day 4, the males and females were split into
experimental and control groups, each group containing 75-100 flies.
These were maintained at 29° C. at 25 flies per vial. All flies
were transferred every two days into fresh media for the first month and
then every day for the following months. The number of dead flies was
counted at each tossing and used to calculate mean and median life spans
for the experimental (+DOX) and control (-DOX) groups. The statistical
significance of the difference in median life span was calculated for
each experiment using log rank tests. Northern analyses. Each of the
indicated hUbbWT(70,80,118), hAppWT(1,24), hUbb+1(1,11) and Oregon R
control strain was crossed to the rtTA(3)E2 driver line (Bieschke et al.
1998) and cultured at 25° C. in urine specimen bottles. Males
containing both the transgene and the rtTA(3)E2 driver were scored and
collected. The males were then split into experimental and control group,
each containing 100 flies. These were maintained at 25° C. at 25
flies per vial. Flies were cultured on plus and minus DOX food for two
weeks, and total RNA was isolated from 30 adult Drosophila males using
the RNAqueous kit (Ambion), fractionated on 1.0% agarose gels and
transferred to

[0160]GeneScreen membranes (DuPont/NEN). 1×=5 μg, and 2×10
μg. The PCR product UBBwt-1 was used as a specific probe for the
hUbbWT gene. The PCR product APPwt-1 was used as a specific probe for the
hAppWT gene. Blots were also hybridized with probe specific for ribosomal
protein gene Rp49 (O'Connell and Rosbash 1984). DNA probes were
32P-labelled using the Prime-It II DNA labeling kit (Stratagene).
Hybridization was carried out in Church-Gilbert solution at 65° C.
overnight. Hybridization signals were visualized and quantified using the
phosphoimager and ImageQuant software (Molecular Dynamics). Developmental
effects of hUbbWT and hUbb+1 overexpression on life span. Flies were
cultured on food supplemented to a final concentration of 640 μg/ml
Doxycycline for the experimental group. Each line of hUbbWT(70,80) and
hUbb+1 (1,11) males were mated to virgins of the "TO-daughterless"
driver described above, in food bottles plus dox or minus DOX. The
progeny were allowed to develop in the plus and minus dox conditions.
Prior to eclosion, bottles were cleared of adult parents and newly
eclosed flies were allowed to emerge over the next 72 hours. The pupae
from both plus and minus DOX bottles were scored for any noticeable
phenotype. At eclosion, all the possible combinations of phenotype were
scored for each cross and condition. Progeny containing both the
transgene and the drivers were screened and collected. Progeny from the
plus DOX bottles were separated into males and females and lifespan assay
was carried out for these two groups on minus DOX vials. Progeny from the
minus DOX bottles were also separated into males and females and put on
both plus DOX and minus DOX vials to generate four groups. Therefore a
total of six groups were generated per line. The flies were transferred
into fresh vials of plus DOX and minus DOX food at 29° C. every
other day for the 1st month and then every day until zero survival.
Molecular misreading reporter lines irradiation treatment assay. MM-ATG
58A, 46A, 34A, 8A, 46B, 8B, MMC-ATG X, and X2 males and females were
cultured for one month on food containing plus dox (640 ug/ml
doxycycline) for the experimental, experimental control and minus dox
(640 ug/ml ampicillin) for the negative control. The negative and
experimental control groups are not exposed to the irradiation. Each of
the experimental lines was irradiated for 9 hours (total 50,000 rads).
The flies were transferred into fresh vials of appropriate plus and minus
DOX food each day until zero survival. At every five days after the
irradiation, each of the experimental and control lines were observed
under a GFP fluorescent microscope (Leica) for GFP expression.

[0161]Molecular misreading reporter lines 100% oxygen survival assay. From
each of the lines MM-ATG 58A, 46A, 34A, 8A, 46B, 8B, MMC-ATG X, and X2,
males and females were cultured for one month on food containing plus DOX
(640 μg/ml DOX) for the experimental. Experimental groups were then
transferred to an enclosed chamber with 100% oxygen gas flow (Landis et
al. 2004). The flies were transferred into fresh vials of appropriate
plus and minus DOX each day until zero survival. Every 24 hrs each of the
experimental and control lines were observed under a GFP fluorescent
microscope (Leica) for GFP expression. Western analyses. Several antibody
reagents were purchased from Upstate cell signaling solutions, including
Anti-App (Catalog #07-667) and Anti-Ubb (Catalog #07-375), as well as
antibody specific for hApp+1 ("Amy-5") and antibody specific for
hUbb+1 ("Ubi2a"), both characterized previously (van Leeuwen et al.
1998). For each of the lines, 30 flies from the experimental group (+DOX)
and 30 flies from the control group (DOX) were collected at 26 days (Time
point 1), 48 days (Time point 2), 67 days (Time point 3), 82 days (Time
point 4) and 105 days (Time point 5) for Western analyses. Thirty adult
flies were homogenized in Laemmli sample buffer (Bio-Rad) and dilutions
were made from the supernatant. The samples were boiled for 10 mins,
cooled and fractionated on SDS-PAGE. Stacking gel was 4% and the running
gel was 12% for the Ubb+1 and 7.5% for the App+1. The samples were
transferred to the nitrocellulose membrane (Bio-Rad) and the membrane was
blocked overnight at 4° C. in PBST supplemented with 5% Non-Fat
Dry Milk (Bio-Rad). Next the nitrocellulose membrane was incubated with
1:2000 of primary antibody specific to Ubb+1 or specific to the
App+1. The antibody diluent was made fresh each time in 1% BSA/PBST
and incubated overnight at 4° C. Horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody (Amersham) was diluted to 1:3000 in
1% BSA/PBST and incubated at room temperature for 2 hours. After washing
steps, the samples were briefly incubated in chemiluminescence reagent
plus (Perkin Elmer) and the bands were detected using Kodak Image
Station. Additional Western control experiments utilized mouse monoclonal
antibody 22c11 (Millipore/Chemicon), specific for the N-terminus of hApp,
and cortical neuron lysates as a positive control for App (data not
shown).

[0162]Although the present invention has been described in terms of
specific exemplary embodiments and examples, it will be appreciated that
the embodiments disclosed herein are for illustrative purposes only and
various modifications and alterations might be made by those skilled in
the art without departing from the spirit and scope of the invention as
set forth in the following claims.